JPH04500004A - High rate of translation of polycistronic messages in eukaryotic cells - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 15. A method for enhancing protein expression in cultured cells derived from multicellular organisms, the method comprising: , polycistronic transcription unit according to any one of claims 1 to 12. into cultured host cells derived from microorganisms, and the cultured host cells are cultured in a suitable medium. A method comprising propagating.

Specification High rate of translation of polycistronic messages in eukaryotic cells Keizu Ridan The present invention relates generally to protein expression, and more particularly to culture media derived from multicellular organisms. Concerning the high rate of translation of polycistronic messages in nutrient cells.

Light■Imperial Family■ Advances in cell culture and recombinant DNA techniques have enabled the use of genetically engineered cells to promoted the expression of various proteins that have value or other economic value. to higher eukaryotic sources Expression of many biologically active therapeutic proteins derived from lower eukaryotic cells is often or require post-translational modifications that do not occur naturally in prokaryotic cells and are therefore highly The use of cells derived from homoeukaryotic sources is required. For example, glycoproteins in mammalian cells White matter expression has the advantage of providing proteins that contain natural glycosylation.

Glycoproteins produced by mammals are superimposed on glycoproteins produced by lower eukaryotes. The outer chain carbohydrates present are very different outer chain carbohydrates. contains. The use of mammalian cells as hosts for the production of secreted proteins is It has significant advantages over secretion from lower eukaryotic cells in this respect. In other words, Mammalian cells readily recognize proteins destined for secretion 4 and process them appropriately. This is not necessarily the case in lower eukaryotes. do not have.

Methods for the expression of cloned DNA sequences in a variety of higher eukaryotic cells are known in the art. It is known as The cloned DNA sequence has been published in the literature (Thilly W., M. ammarian Ce1l Technolob-Butterworth Puhlishers, 5tonehaa+, MA) into mammalian cells using methods described. Birds (Kretsoval) i et al. Gene, 58: 167-176, 1987), insects (Miyaj ima et al., Gene, 58: 273-282.1987) and fish (I sa and Shima, Mu Ce11. Sci., 88: 219-224.1 Cell lines from other organisms can also be used, including cell lines from 987). Wear. In mammalian cells, expression of cloned DNA sequences is controlled by promoter sequences. sequence, enhancer sequence, leader sequence, splice signal and polyazonylene Expression units that contain transcriptional control signals, including has been enhanced by inserting quality coding sequences. transfected Identification of clones containing the selected DNA sequence is achieved by adding a selection marker along with the expression unit. This is facilitated by simultaneous introduction.

Optimize expression levels by amplification, e.g., using a selection of amplifiable selectable markers. can be converted into However, simultaneous amplification of expression units is particularly important for selective markers. If the car is introduced as an independent DNA sequence, in all clones It's not guaranteed.

Insertion of a cloned DNA sequence into an expression unit allows the expression unit to be inserted into a host cell. does not guarantee effective gene expression when introduced. Cloning code sequence Low expression levels may be due to inefficient transcription or translation of the coding sequence or unstable mechanisms. NGER DNA (mRNA) sequence, protein production instability, or expression vector This may be caused by the presence of toxic sequences in the cormorant. Recombinant proteins may be imprecisely, inappropriately, or inefficiently processed by host cells into post-transcriptional proteins. There is a possibility that it will be processed.

Efficient expression of coding sequences in eukaryotic hosts also requires expression of associated proteins. However, this protein requires processing, stabilization or would require modification. Optimal expression of biologically active recombinant proteins also requires It will depend on the presence of translation and/or transcription factors. These proteins are recombinant proteins. It will be present in host cells at such low levels that efficient expression in white matter is limited.

Examples of proteins that require specific post-translational modifications include certain clotting factors; These factors require T-carboxylation of specific glutamic acid residues for biological activity. β-hydrosomes of certain aspartic acid residues are required and further biologically active. May require conversion to xiaspartate.

Some proteins exist as multimers in their active form , some of which are composed of distinct subunits. some kind of mulch mer proteins, such as insulin, are encoded within the same cistron; and post-translationally processed into multimers containing heterogeneous polypeptides. . Other multimeric proteins are cocooned by DNA sequences that are not located within the same cistron. is coded. Examples of this type of fluorescent matter include tetramers made from a and b chains. Confounding factor X■, PDGF existing as an A-B dimer, immunoglobulin , hemoglobin, and major histocompatibility antigen. within the same cistron Expression of non-encoded multimeric proteins has not been reported for immunoglobulins. (Hickman and Kornfield, J. Immunol. 121: 990-996.1978; Kearney et al., Monoclo nal Antibodies and T-Cell Hbridomas; Hammerling, Hammerling and KearneykJa collection, Elsevier/Northland Biomedical Press+ 379-387, 1981;) Iendershot et al., J. Ce1l Biol, 104: 461-767, 1987), the same for secretion. Co-expression of all subunits in the same cell or subunits to ensure biological activity. Achieving correct assembly into the proper placement of the unit will require .

To compensate for the problems inherent in the expression of cloned coding sequences, cloned DNA sequences Other sequences can be introduced into the cell that function to enhance or enable expression of the sequence. Often advantageous.

These other DNA sequences include processing enzymes, transcription factors, translation factors, and proteins. Coding sequences for stabilization of and protease inhibitor substances are included. host cell Optimal expression of recombinant proteins in proteins requires the simultaneous introduction of many coding sequences. Probably.

Methods for introducing multiple expression units into host cells include the use of multiple expression vectors. Co-transfection (Dubois et al., Proc, Natl, Acad, Sci, tlSA, 77: 4549-4553+ 1980; Subr. amani and Berg, Cedan, 16: 777-785.1979) , transfection with a vector containing multiple expression units (Staf Ford and Queen, Nature, 306: 77-79.198 0 ; 0chi et al., Proc, Natl.

Acad, Sci, USA, 80: 6351-6355.1983; Kadesch and Berg+Mo1. Ce11. Biol, +dan: 259 3-2601.1986) and vectors containing polycistronic transcription units. transfection with a transfection agent (Peabody and Berg, Mo1.C e11. Biol, +dan: 2695-2703゜1986; Kaufma n et al., EMBOJ, 6: 187-197. 1987 HBoel et al. , FEBS Lett, , 219: 181-188.1987; Le vinson and Simonsen.

U.S. Pat. No. 4,713.339). However, in reality, this Their method has been shown to have serious limitations.

The major limitation to co-transformation of multiple expression vectors is the commonly used selection The number of markers is limited. These selection markers are dominant markers - and non-dominant markers (for cell lines lacking activity compensated for by selectable markers). and those that confer compensatory activity). The selection of the optimal selection marker system depends on the This is because there are few selectable markers that have been shown to co-amplify DNA sequences that Ru. Selectable markers are reviewed by Thlly (supra).

Introduction of multiple expression vectors results in multiple deficiencies in activity compensated by selectable markers. or the compound to which the selectable marker confers resistance. Post-translational processing that is highly sensitive and required by many recombinant proteins It is also limited by the number of useful cell lines known to provide coverage.

Identification of cell lines that are considered suitable hosts for specific selection and expression systems 'ensure that the cell line is amenable to marker selection or amplification. do not have. Mammals are capable of gene amplification in lower eukaryotic and prokaryotic cells. This is not possible in cell lines with multiple marker deficiencies or susceptibilities. requires extensive screening to identify cell lines that do. Therefore, there is A large number of selective systems are available for transfection of cells with multiple expression units. There are certain restrictions.

The possibility of simultaneous introduction of multiple expression vectors into host cells depends on the DNA sequence to be introduced. decreases with increasing number of columns. Simultaneous increase of expression units in selected clones The width is also an unexpected phenomenon. All simultaneous trials for the same amplified gene dose The possibility of simultaneous amplification of transfected DNA sequences depends on the number of DNA sequences involved. Both decrease. All transfected strains at approximately the same gene dosage. Identify co-amplified and co-transfected clones containing the current vector. Extensive and expensive screening methods would be required to determine the outcome.

As mentioned above, expression vectors containing multiple expression units have not been reported in the literature. (Stafford and Queen, supra; Och et al., supra; L au and Kans, supra; Kadesch and Berg, supra). however , these constructs carry only 2 or 3 expression units and One expression unit contains a selection marker. More than 3 expression units Although it is theoretically possible to create vectors containing Not reported during the dedication. In reality, the production of such vectors is complex and It's a waste of time.

suggesting that optimal expression depends on the orientation of the expression unit present on the vector. The results of Kadesch and Berg (supra) suggest that in order to avoid transcriptional interference, Generation of vectors containing multiple transcription units by requiring directional restriction make it even more difficult. Transfection using this type of vector is recombinant ( recon+bination or rearrangement) phenomenon. This phenomenon may result in the exclusion of certain expression units present on the vector. may result in Identification of clones in which all expression units are active is widespread. would require extensive and expensive screening.

Recent reports of genetically engineered polycistronic transcription from a single promoter The possibility of expression of multiple coding sequences (cistrons) was proposed. However, this Expression of downstream cistrons in these genetically engineered polycistrons was limited. It brought only success. Polycistronic transcription unit carried on a plasmid (Peabody and Bergs supra; Kaufm an et al., supra HBoel et al., supra; Levinson and Simonsen, supra. U.S. Patent No. 024,713,339), but from polycistronic mRNA The level of translation of the downstream cistron was shown to be dramatically reduced.

Downstream cysts shown in currently reported polycistronic transcription units Severe suppression of Ron's expression makes this system susceptible to high-level expression of numerous proteins in eukaryotic cells. This makes it unworkable. Multiple expression vectors for expression of multiple proteins Co-transformation with vectors reduces the number of available selection systems and associated cell lines. Expression of multiple proteins from a single vector is not feasible because , is cumbersome and difficult due to constraints in the production of such vectors.

Therefore, means are available in the art to increase the expression of numerous proteins in higher eukaryotic cells. It is necessary.

Lord Yu■ Saradama In summary, the present invention provides methods for enhancing protein expression in cultured cells derived from multicellular organisms. Disclose how to do so. This method generally uses (a) the following equation %formula%) (In the formula, P is a transcription promoter; C is a DNA sequence encoding a protein; HEL is a high efficiency leader; n is a positive integer greater than 0; and m is an integer from 1 to 8) introducing into cultured cells a polycistronic transcription unit represented by; and (b) growing the cultured cells in a suitable medium;

In a preferred embodiment, the cultured host cell is a mammalian host cell capable of producing L. Will Three ticks, especially high efficiency virus readers, are preferred. In one aspect of the invention C7 and C7 are factor X, platelet-derived growth factor, and immunoglobulin. or be a subunit of a multi-subunit protein such as a histocompatibility antigen. Can be done.

Polycistronic transcription units and such polycistronic transcription units for use in the aforementioned methods Also discloses cultured cells derived from multicellular organisms into which polycistronic transcription units have been introduced. be done.

These and other aspects of the invention will be apparent from the following detailed description with reference to the accompanying drawings. It will become clearer.

Page 2 Figure 1 shows a vector containing a polycistronic transcription unit in vector pML-1. Figure 2 shows the production of lasmid pBoe1360. The symbol used is Ad5 ori : O-1 map unit of adenovirus 5; E: SV40 enhancer Sequence, Ad2 MLP: major late promoter from adenovirus 2; Ll -3: Adenovirus 2 tripartite leader sequence ; 5'ss: 5' splicing signal; 3'ss: 3' splicing signal and pA: Late polyazonylation signal from SV40. Ru.

Figure 2 shows the construction of plasmid pTP/C1a.

FIG. 3 shows the production of nidistrone mRNA. Figure 3A is plasmid pD5 mRNA produced by cells transfected with CAT-DHPR' Analysis of A (arrow) is shown. The numbers on the right relate to size markers. B is 2 cis Diagram of tronic expression unit, bicistronic mRNA and probe. show.

Dotted lines indicate DNA that is apliced out of some transcripts.

Figure 4 shows the construction of plasmid pTP/F9/C1a.

Figure 5 shows the construction of plasmid pFVn-P-BiP.

■No for Before describing the present invention in further detail, it is understood that the terms used below are defined. will be of help.

Sisteron A protein is a DNA sequence that encodes a polypeptide. This DNA sequence is a gene, cD It can be in the form of NA, or a synthetic NA fragment, or a clone thereof.

fA” Ji Gan Toji Z A DNA sequence containing at least two cistrons, the cistrons at least the stop codon of the upstream cistron and the translation start codon of the downstream cistron. separated by a Polycistrons are functional transcriptional proteins between cistrons. Contains no motor.

No.21 plate base Translation stop codon in the upstream cistron and translation start codon in the downstream cistron in a polycistrone. DNA sequence between the start codon and the start codon.

Polycistron − Unit DNA sequence containing a polycistrone operably linked to a transcription promoter . Transcription of a polycistrone is a single-mRN containing sequences corresponding to the constituent cistrons. bring about A.

Uni l two dishes A 5'-untranslated region that performs efficient translation of transcribed messages.

Engineering and Era Δno” Jin Otsu Kuto mRNA splicing events (i.e., cleavage and ligation reactions that lead to excision of intervening sequences) Mount (Nuc, Ac1ds), which has been functionally shown to be involved in Res., 10: 459-472.1982). is a sequence that shows commonality with the 3' sequence.

As summarized above, the present invention provides a method for controlling downstream cysts in polycistronic transcription units. Novel NA constructs useful for increasing the level of protein expression from Ron disclose something. These novel NA constructs contain polycistrons and this port In the lycistron, multiple DNA sequences encoding multiple proteins of interest are arranged vertically. DNA sequences that are linked in tandem and encode a leader sequence Separated by These polycistrons are highly potent in appropriate expression vectors. transcriptional and translational signals are linked and cultured from multicellular organisms. introduced into cells. Surprisingly, these constructs inhibit the expression of downstream cistrons. It was found that it resulted in an increase.

The polycistrons of the present invention coexist in such a way that the leader sequence is present between the coding sequences. It can be formed by linking a code sequence to a leader sequence. each code The sequence must have its associated translation initiation and termination signals in the correct leadership position. within the framework. In a preferred embodiment, the leader sequence is Inserted into the intercistronic region using restriction endonuclease digestion and ligation.

Preferred leader sequences are viral leader sequences, including adenovirus First leader and adenovirus L, -IX leader (Berkner and 5h arp, Nuc, Ac1ds Res, 13: 841-857+19 85), 5V401J-der and parvovirus Includes:

A particularly preferred leader sequence is a highly efficient viral leader, adenovirus trypa adenovirus tripartite leader (Ll- 3). Suitable cellular leaders include the ovalpmin leader. Lee It is advantageous to add the reader sequence directly to the translation initiation sequence. One aspect of the present invention In the transcription unit, the leader sequence separates the splice from the splice signal within the transcription unit. It is added to the translation initiation codon after the lysing event. In other embodiments, the lead The primer sequence is linked to the translation initiation sequence by in vitro mutagenesis.

This strong translational signal in the intercistronic region of polycistronic transcription units Cultures derived from multicellular organisms such as mammalian cells infected with recombinant viruses containing cultivated cells can produce levels comparable to those obtained using monocistronic expression units. It was found that the downstream cistron was expressed in the cell. Inter-cistron leader failure In its presence, the downstream cistron is not detectably expressed in infected cells. Sith A plasmid carrying a polycistronic expression unit containing an intertron leader More transfected cells have comparable cells lacking intercistronic signals. At least a 20-fold increase in protein expression when compared to cells containing Sumid shows.

Suitable leader sequences include 5'-non-coding sequences for viral or plasmid test systems. It can be identified by the insertion of

Such systems have been used to study adenovirus leader sequences (e.g. For example, Berkner and 5harp+ Nuc, Ac1ds Res, +13 : 841-857.1985, and Kaufman, Proc, Nat. l, Acad, Sci.

(See Otoko Sei, 82: 689-693.1985). In summary, note The eye potential leader sequence (5'-non-coding sequence) acts on the marker coding sequence. inserted into an expression unit comprising at least a transcription promoter operably linked to so that the potential leader sequence is directly 5' to the transcription start site of the marker coding sequence. be inserted directly. The marker code sequence is preferably a measurement method for exists. Marker coding sequences include DHPR and hepatitis 8 surface antigen ( Davis et al., Proc. Natl. Acad.

Sci, USA, 82: 7560-7564.1985).

Leader sequences of interest for testing potential leader sequences in plasmid systems higher eukaryotic cells can be transfected with expression units containing Insert into vector.

Vectors suitable for transfecting mammalian cells include pBR322 ( Derivatives of Boliver et al., Gene, 2: 95-113.1977), For example, pML I (Lusky and Botchan, Nature, 29 3: 79-81+1981), and ptl (Messing, Meth, Enz mol, 101: 20-79.1983) Vector derivatives Included. A suitable vector for use in transfecting other host cells. For example, Kretsovali et al. (supra), Miyajima et al. Isa and Shima (supra). emanation The current vector is transfected into host cells, and the expression of the marker is determined by - expressed by cells transfected with an expression vector that does not have the Compare with the marker. Transduced using an expression vector without a leader sequence. Transfected cells are transfected with an expression vector containing a leader sequence. At least a 5-fold increase in marker expression in transfected cells indicates a suitable lead. identify the sequence. 5 times more than expression by expression vector with leader sequence Large increases identify highly efficient leaders. The leader sequence is similar to adenovirus. In the virus system, the recombinant virus is produced using the plasmid expression unit described above. It can be tested by making it. host using the resulting Mi recombinant virus. Cells are infected and the level of marker expression is determined. leader array Contains a leader sequence for cells infected by expression units that do not have at least 5 times the expression of the marker in cells infected by the expression unit. Increase identification of suitable leader sequences. Expression unit with leader sequence A greater than 5-fold increase in expression of the marker identified as a high efficiency leader sequence. appropriate Once appropriate leaders have been identified, they can be used to investigate polycistronic development, as described above. Create the current vector. Such vectors have additional components that promote high-level expression of interest. It will contain additional genetic elements.

The polycistronic expression unit of the present invention is used as a vector for the production of proteins of interest. Insert into. Suitable vectors include the recombinant plasmids described above. recombinant will Vectors include SV40 viral vectors and adenoviral vectors. (For an overview see Thllly, supra). Particularly preferred vectors are recombinant It is an adenovirus (hereinafter referred to as rAdJ) vector. Using Ad vector The expression of cDNA for each cDNA is described, for example, by Berkner et al. , 13: 8417857.1985). politics Recombinant adenoviral vectors containing tronic transcription units can be used in any tissue or have the advantage of introducing transcription units into virtually all cells of a cell line. do. When polycistronic transcription units should be used in gene therapy , which is particularly advantageous.

Polycistronic containing one or more DNA sequences encoding the protein of interest To obtain expression of the transcription unit, the vector will usually contain additional elements. . The transcriptional promoter is located upstream of the translation initiation signal of the first cistron.

Suitable promoters include the mouse metallothionein (MT-1) promoter. -(Palmiter et al., 5science, 222: 809-814゜1 983), SV40 late promoter (Piatak et al., J. Virol,. 48:503-520.1983), SV40 late promoter (Beno ist and Chambon.

290:304-310.1981) and cytomegalovirus (CMV) promoter. Viral promoters direct transcription preferred for their efficiency. Any effective program in the disclosed method Although promoters can be used, particularly preferred promoters are adenovirus promoters. It is the major late promoter from the expression unit , a leader sequence located downstream of the promoter and upstream of the first cistron. It would be preferable to contain. Preferred leader sequences include adenovirus Lus first leader, Adenovirus L1-IX leader and Ovalbuminley Includes: A particularly preferred leader sequence is adenovirus tripartite. ・Be a leader. Suitable leader sequences are identified by the screening method described above. can do. The expression unit has a 5'-splice signal and a 3'-splice signal. The splice signal may contain a splice signal consisting of an is signal.

The 5'-splice signal may be associated with a leader sequence such as any of the above. I can do it. In a preferred embodiment, the 5'-splice signal is an adenovirus. This is a sequence related to the ST3 leader. The 3'-splice signal is rabbit ・β-globin 3'-splice signal (Ruskin et al., Kikitan.

38:317-331.1984). For viewing purposes, see Mount, supra). Special Preferred 3'-splice signals are those from the variable regions of immunoglobulin genes. It is something. Furthermore, the expression unit contains a DNA sequence containing a polycistrone. A polyazonylation signal located downstream from the column is included.

Viral polyazonylation signals, e.g. early or late from SV40 polyadenylasidine signal or polyadenylasidine signal from the adenovirus E1b6ff region. Azonylation signals are particularly preferred. Polyazonylation signal It can also be supplied by coding sequences present in polycistrons.

Preferred vectors further include an S Enhancer sequences such as hangers can be included.

In some cases, selectable markers are introduced into cells together with polycistronic transcription units. Preferably, the 0 selection marker is a neomycin resistance gene, a hygroma Isin gene, Ecogp gene, thymidine kinase gene, adenine phosphorus Holibosyltransferase gene, hypoxanthine phosphoribosyltransferase ferase gene, and multidrug resistance factors (Roninson et al., Proc, Nat. l, Acad, Sci, υ5A, 83: 4538-4542.1986 ; Lleda et al., J. Biol, Ches., 262: 505-508 ．． 1987). Preferably, the selectable marker is an amplifiable selectable marker. It is. A preferred amplifiable selectable marker is the DHFR gene. Especially preferred A highly amplifiable selectable marker is DHPR' cDNA (Simonsen and L. evinson+ Proc, Natl, 8cad, sci, UsA, Dan0: 　2495-2499. 1983). The selection marker is Th1lly ( (ibid.) and the selection of selectable markers is within the level of those skilled in the art. It is within the range.

The selectable marker is a separate DNA sequence at the same time as the polycistronic transcription unit. as a cistron in a polycistronic transcription unit or in the same vector. can be introduced into cells as an independent expression unit. Amplifiable selection The marker is placed in a polycistronic transcription unit as a terminal cistron, and this The selection marker and the second cistron from the end are separated by the leader sequence. It is particularly preferable to prevent this from happening. A selection marker placed in this way will decrease The selectable marker and and thereby force faster amplification of DNA sequences.

The polycistronic transcription unit described in this invention can be used in recombinant proteins in eukaryotic cells. It has wide applications in the production of white matter.

These uses include producing economical quantities of therapeutically or economically important proteins. of polycistronic messages to be used in clinical or gene therapy applications. Includes use. The polycistronic message encodes the protein of interest and encodes a sequence that enhances or enables expression of the biologically active protein of interest. It can be adapted as follows. to enhance and enable recombinant protein expression. The proteins that function for this purpose include processing enzymes, protease inhibitors, and stabilizing factors. children, transcription factors, translation factors and selectable markers.

As mentioned above, polycistronic transcription units are used in gene therapy to It can be used in combination with the rus vector system. The gene therapy used here is the insertion into an organism of a DNA coding sequence that corrects a genetic defect in the host organism. Ru. Currently, the human tissues used for gene transfer are bone marrow and skin cells. be. Effective expression of the protein of interest in tissues where it does not naturally exist Polycistronic transcription units can be created that allow for. human remains Today's research into gene therapy involves introducing it into human cells by insertion into retroviruses. Concentrates on the use of human genes to be used. In adenovirus vectors, Promoters or boriadenyl promoters as observed in torovirus vectors Inhibition by intron signals or recombination of intron-containing genes (rearrange) Ment) has not been reported, so for gene introduction for gene therapy. Adenovirus vectors may have advantages over retrovirus vectors. a Denoviruses can transform various human and murine cell lines to produce stable integrants (i.e. It is possible to form a Has a proven ability to enter all cells during staining.

Therefore, highly infectious and transforming recombinant adenoviral vectors are useful for gene therapy. Therefore, the advantages over retroviral vectors will be clear.

Therapeutically useful proteins and proteins that can be expressed in polycistronic transcription units DNA sequences encoding economically useful proteins include those encoding blood clotting factors. those encoding various serine proteases, those encoding growth factors , protein C encoding, protein S encoding, tissue plus Those encoding minogen activators, those encoding immunoglobulins, and those encoding anti-inflammatory factors. - those encoding inflammatory proteins, those encoding anticoagulants and analogues; including, but not limited to, those encoding derivatives of these proteins. stomach.

Many of these proteins exist as multi-subunit proteins, and their coding The sequences are not located within the same cistron. Multi-subunit proteins in mammalian cells Quality expression requires expression of the coding sequences for all of the subunits. Many Subunit proteins include factor Xm, platelet-derived growth factor, and immunoglobulin This includes hemoglobin, major histocompatibility antigen, and hemoglobin. components within the same host cell Facillus is an example of a multi-subunit protein that requires co-expression of subunits. and immunoglobulins. Factor X is a2 dimer and b2 dimer (Chung et al., J. Biol., C. hem, 249: 940-950.1974). b2 dimer is a2 da It was shown to function to stabilize the imer. Immunoglobulins are functional immune The secretion of epiglobulin requires the expression of both heavy and light chains. It is known that (Hickman, l and Kornfield, supra; K6 Arney et al., supra;) Tendershot et al., supra.

Polycistronic messages also efficiently release high levels of biologically active proteins. processes proteins that are required at enhanced levels to produce can be decoded. Processing proteins involve opening precursor proteins at specific sites. a protease that is cleaved to yield a protein or proprotein in the adult μ) form and/or in the active form; enzymes, or proteases that cleave single-chain proteins into multi-product forms. process Other examples of modifying proteins include proteins that modify proteins, such as T-carboxylase. , specific glutamic acid residues in certain clotting factors and other calcium-binding proteins. Modifying enzyme; asparagus, a necessary modification for the biological activity of protein C Enzyme responsible for converting gic acid to β-hydroxyaspartic acid; as well as purine It is an enzyme responsible for hydroxylating residues. Other processing proteins include milli Stoylation, C-terminal amino acid removal, sulfation, C-terminal amidation, and glycoprotein These are proteins responsible for the addition of carbohydrate chains. Naturally found in mammalian cells Processing enzymes that are absent and can be introduced into polycistronic transcription units An example is S6 cerevisiae (S, cerevisiae) ■1 gene product It is. The gene product is a cleaved endopeptidase at the Lys-Arg residue. It is For example, the silicon 11 coding sequence and the protein C coding sequence are Encoded in a tronic transcription unit. The KEXλ gene product is a precursor form of the protein. Promotes Lotin C processing. 1st-cistron to promote secretion It is preferable to place a sequence encoding protein C, a secreted protein, at this position. Delicious.

The polycistronic transversion unit also carries coding sequences to stabilize proteins. can be included. Stabilizing proteins block hydrolytic destruction of the protein of interest. A protease inhibitor that binds to the protein of interest and protects it from degrading enzymes. protein that binds to the protein as a cofactor, or that is required by the protein. and other molecules that inactivate cofactors. polycistrone Examples of coding sequences that function to stabilize or promote activation of other fluorescent substances in is that of protein S. Protein S is protein C, i.e. protein The protein C-protein S polycistrone improves the expression of activated protein C. There will be. An example of a stabilizing protein is von Willeplant (von Willeb). rand) factor (v14F). , codes for both vWF and coagulation factor ■ polycistrons produce wWF, which functions to stabilize clotting factors, and will prolong the half-life of Factor II in the extracellular medium.

Polycistronic transcription units include transcription and translation factors (D)'nan and Trj an, Nature, Todoroki: 774-777, 1985; Baeurl e and Baltjmore, Cedan, 53: 211-217.1988 ;) Lattman et al., for buttons.

55: 345-35L 1987; Kaufman et al., Mo1. Ce1 1. Biol, 7: 3759-3766, 1987) A sequence may be included.

Introduction of these factors disrupts endogenous cellular machinery to provide more efficient gene expression. Supplementing the structure can lead to better expression. cloned Examples of transcription factors that are promoter-specific31)1 (Dynan and Tijan , supra). encodes or appears to encode an spl recognition sequence The gene of interest that has been modified is the spl nodame coding region linked to the cDNA. polycistronic transcription units comprising regions that contain more of the coding sequence of interest. will enable transcription.

polycistronic transcription sequences that promote the secretion of other proteins. It is preferable to include it in the knit.

It encodes a protein that can help secrete fluorescent substances and other proteins. Examples of polycistronic transcription units that include the coding sequences of Factor ■ and BiP It contains.

The immunoglobulin binding protein BiP is found within the cavity of the endoplasmic reticulum (ER) and is associated with C - having the terminal amino acid sequence Lys-Asp-Glu-Leu (Munro and Pelham+ Ce11. Average: 291-300.198'a). Munr.

and Pelha'm (supra) showed that this tetrapeptide KDEj,... We are focusing on the fact that this is part of the signal that causes protein retention. BiP may be hindered due to some homology with the C-terminal amino acid sequence of . centre The coexistence of the factor “■” and BiP, which is mainly maintained in the ER, results in the maintenance of the host ER. saturates the system and therefore allows Factor■ to be secreted more efficiently Will.

Expression vectors of the invention can be used, for example, for calcium phosphate-mediated transfection. (Wigler et al., Ce1l, 14: 724.1975 Corsar o and Pearson, Somatic Ce1l Genetics, 7 : 603+ 1981; Graham and Van der Eb, ν1ro 1. ．． 52: 456-467, 1973) or by electroporation. (electroporation) (Neuo+ann.

It can be introduced into cultured cells according to Condom, 1: 841-845.1982). can. Viral expression vectors can also be used, for example, by Kaufman (Proc, Na tl, 8cad, sci, 1JsA, 4ξj2: 689-693.1985 ) can be used to infect host cells using the method described by can. Addition of an additional substance known as "carrier DNA" to the mixture introduced into the cells It is also preferable to add DNA.

Cultured cells derived from various higher eukaryotic host cells or multicellular organisms can be used in the present invention. can be done. DNA sequences that can be propagated in culture and cloned Cells capable of expressing ), amphibians (Illolf and umby, 5science+ 144: 1571L 1964; and Freed et al., Biology of A+ nphibian Tumors, Mizellg, pp. 101-111, S pringer Verlay+ 1969), Earth (C1ark, J, N atl, Cancer1968 i and Kretsovali et al., Gene. 58: 167-176, 1987). Use in the invention Preferred mammalian cell lines for include C05 (ATCC CRL-1650), BHK (ATCC CLL 10), CIO and 293 (ATCC1573) each cell line, as well as derivatives and isolates of these cell lines. ), but other cell lines may be preferred for production of specific proteins. It will be clear to those skilled in the art. Mammalian tissues are also suitable for use in the present invention. be. Generally, the host cell line or tissue produces high levels of the protein of interest. based on its capabilities and/or its suitability for use with a desired selection marker. will be selected accordingly. However, the present invention provides substantial allows production of virtually any protein in any cell line do.

After introducing the expression vector into a host cell, the cells are generally incubated for a period of time, typically 1 to The cells are grown for 2 days to start expressing the gene of interest. Contains the expression vector of the present invention The cells are grown in a suitable medium. Charcoal is a medium that promotes the growth of cultured eukaryotic cells. natural sources, amino acids and vitamins in a normal diet supplemented with defined growth factors and serum. Contained in balanced salt solution. Selection of appropriate media to optimize cell growth is within the skill of the art. and depending on the specific characteristics of the tissue or cell line to be grown. Dependent. Media formulations are well known in the art (e.g. Mamma lion Ce1l Curture: The Use of 5erur a-FreeHora+one-Seppied Media, M ather collection, P1enua+ Press-.

New York, NY, 1984; Th1lly, supra; and ATCC Kata (see log) and can be done according to published formulation examples or commercially available. (e.g., Gibco-Life Technolo gies Incl Lawrence, MA; ^TCC, Rockville e.

during) selective pressure is applied to select for growth expressing the 0 selection marker. can do. For example, methotrexate When using a selection method, stepwise increasing drug concentrations are used to increase the number of copies of the coding sequence. Allows selection and results in elevated expression levels. Inject a clone of such cells. Eye protein production can be screened. cause cells to proliferate , and isolate the protein from the cells by cell lysis. Useful screening methods Includes immunoassays and activity assays.

Methods for purifying recombinant proteins are generally known in the art. Pallid matter is host cell If the cells are to be kept in a It is necessary to obtain a clear cell lysate by removing debris. secreted If the protein is purified directly from the culture medium, the protein is purified directly from the culture medium. Clear cell lysate or medium is fractionated using conventional protein purification methods. Generally a multi-stage method will be used. to this Typical methods for this include precipitation (e.g. polyethylene glycol or ammonium sulfate). monium precipitation), ion exchange chromatography, affinity chromatography chromatography, preparative gel electrophoresis, high performance liquid chromatography, and This includes pressure, pressure, and liquid chromatography. In some cases, the stage removal of excess salt by e.g. ammonium sulfate precipitation followed by dialysis. It is preferable to U the fraction of interest.

The selection and order of the various steps will depend on the properties of the particular protein of interest and will be within the skill of those skilled in the art. It would be within the range of

The following example is illustrative and not limiting.

Anal Starry Summer I Ad5 ori, SV40 enhancer-1Ad2 major late promoter, Ad 2) Rivaltite leader, 5' and 3' splice signals, DHFRcDN A 5SV40 polyazonylesion signal and pnt, -i vector sequence A plasmid pD3 was created containing a unique B A cl1 site was introduced. To create plasmid pD3, pDHFRI[[ 0f in the middle (converting the Pst1 site immediately downstream from the FR coding sequence to the Bcl1 site) did. The cohesive end of pDHFRIII partially digested with PstI was converted into T, DNA Removed using dCTP in the presence of polymerase. Next, the DNA was dipped in ethanol. ligated to a precipitated and kinased Bcl I linker. resulting connection The mixture was digested with BclI and electrophoresed in an agarose gel. 5 ．． The 8 kb fragment was isolated from the gel and recircularized by self-ligation. desired modification A plasmid with the following was selected and named pDHFR'. plasmid p DHFR' was transformed into dAM-E. coli strain and plasmid DNA was prepared. Ta.

Next, pDHFR' and psV40 (pML Bawl I-digested 5V40 DNA cloned into the Baa+81 site of -1 ) was digested with Ba1l and Ba+sHI to generate 0.2 kb SV40. The rearenylasin signal and the 4.9 kb pDHFR' fragment were isolated. Next, the 0.2 kb psV40 fragment and the 4.9 kb pDHFR' fragment were linked. The plasmid pD2' was constructed.

“Potson” sequences (Lusky and Bottc) in the pBR322 region Han.

Nature, 293: 79-81.1981). The lasmid pD2' was modified. Plasmids pD2' and pML-1 were transformed into ECORI and NruI, and the fragments were separated by agarose gel electrophoresis. did. Isolation of 1.9 kb I) D2' fragment and 1.8 kb pML-1 fragment and then - concatenated together. Select a plasmid with the desired structure and p It was named D2. Next, this plasmid was digested with EcoRI and BgllI, and the 3' splice signal, the SV40 polyazonylation signal and the p A 2.8 kb fragment comprising the l'lL-1 vector sequence was isolated and the fragment It was called C.

To generate the remaining fragment used in the construction of pD3, pDHFRI[[ was modified to convert the 5stII site into a former ndn[ site or Kpn1 site. P The lasmid pDHFRI [[ was digested with 5stII, dCTP and T, DNA Incubated with polymerase and kinased old ndII [ linker or Kpn I linker. If the resulting plasmid is Digested with old ndIII or KpnI and then electrophoresed through agarose. did. Next, the gel isolated DNA was ligated and transformed into colon WRRI. It was used to The resulting plasmid was transformed into pDHFRII [(HindI[l) and It was named pDHFRm (KpnI). Next, 700kb Kpn ■- Bgl II fragment (fragment A) was extracted from pDHFRm (Kpn I). By digestion with If and Kpn I followed by agarose gel electrophoresis. Purified.

The SV40 enhancer sequence was converted into pDHFRI[I (formerly ndllI) as follows. inserted into. 5V45 DNA was digested with old ndnl and old ndl [cSV 40 fragment (5089-968 bp) was gel purified and pDHFRII [(H It was inserted into the old ndI[I site of indI[I]. Next, the resulting plasmid pE2 was digested with EcoRI and KpnI, and then digested with Ad5 ori and SVO enhancer. A 700 bl) fragment containing the sensor (designated fragment B) was isolated.

For the final construction step of pD3, fragments A, B and C were ligated by a ternary ligation and The mixture was used to transform E. coli RRI. Select a positive clone and This vector was named pD3.

f! ! ! L2LD5 and Dll's j Ad5 ori, SV40 enhancer-1Ad2 major late promoter, hed 2 tripartite leader (Ll-3), 5' and 3' splice signals, S Contains the V40 polyazonylation signal and pML-1 vector sequence. We created plasmids ρD5 and PDII and inserted a unique BamH1 site. . pDHF partially digested with Pstl to generate pD5 and poll Incubating RIII with dCTP in the presence of T, I) NA polymerase By converting the Pst1 site immediately upstream of the DHFR sequence to the BaIIIB1 site pDtlFRIII was first transformed by converting into pDtlFRIII. Next, evaporate the DNA Knoll precipitated and ligated to a kinased BamHI linker. Ba By digestion with a+HI followed by gel electrophoresis and isolation of the 4.9 kb fragment. The excess linker was removed. The 4.9 kb fragment was recircularized by self-ligation. eye A plasmid with the targeted modification was selected and named 9mouth1'.

Next, in order to form plasmid pD1 from pD1', first pVs40 (pM BamHI-digested SV40 cloned into the Bawll 1 site of L-1 (comprising DNA) was digested with Bcl I and Baa + HI to 0.2 k b. SV40 polyazonylesion signal was isolated.

Plasmid pD1' was linearized by digestion with BaIIll and 4 ．． A 9kb fragment was isolated. These fragments were then coated with SV40 polyazony resin. Plasmid pD1 was created by ligating in the backward direction with the column.

“Poison” sequences (Lusky and Bo) in the pBR322sff region tchan.

Nature 293: 79-81.1981). Sumid pD1 was modified. Plasmids pD1 and pML-1 (Lusky and Botchan (supra) was digested with EcoRI and NruI and cleaved. Pieces were separated by agarose gel electrophoresis. 1.9 kb pD1 fragment and 1. The 8 kb pML-1 fragment was isolated and ligated together. have the desired structure A plasmid was selected and named ppD1. Then this plasmid was digested with EcoRI and BglII and the 3' splice signal, S Comprising the V40 polyazonylation signal and pML-1 vector sequences A 2.8 kb fragment was isolated and designated fragment C'.

The SV40 enhancer sequence was converted into pDHFRnl (BindI[[) inserted into. SV40 DNA was digested with old ndll and Hindll [The CSV40 fragment (5089-968 bp) was gel purified and p DHFRI[[(Hindul) was inserted into the old ndI[[ site. Concatenation mixture It was inserted into E. coli RRI. Plasmid DNA prepared from the transformant is digested with restriction enzymes. Screening was performed by elementary analysis. Two plasmids were isolated. One of them is p The enha directed at Kpn I, which is called H1 and is far from Ad5 ori. and the other is called pH2, close to Ad5 ori. It contained an enhancer oriented with I. Next, plasmid pH1 was Digested with coRI and KpnI and digested with Ad5 ori and SV40 enzymes. A 7oobp fragment containing the hanger (designated fragment B') was isolated. plus MidopE2 was digested with KpnI and BglII and the 0.9kb fragment ( Fragment D') was isolated.

The remaining fragments used to create pDII and pD5 were pDHFRm ( Kpn I). Plasmid pDHFRIII (Kpn I) (Example 1) was digested with Kpn I and Bgl II to produce 700 bp Kpn I -BglII fragment (designated fragment A') and EcoRI and Kpn A 0,4 kblr fragment containing Ad5 ori (fragment E' ) was isolated.

Three-way ligation of fragments A', B' and C' for the final production step of pD5 and pDll. and fragments C', D' and E' were ligated in a ternary ligation. Then the mixture was transformed into E. coli RRI. Plasmid from ligation of fragments A', B' and C' The pD5 was designated as pD5 (Fig. 1). Plasmid from ligation of fragments C', D' and E' The cell was designated pDll (Fig. 4).

Flood Boe1360 and d5 (CAT-DHPR) l + Mouse DHPR' cDNA and a polycistronic transcription unit containing the CAT gene. plasmid pBoe1360 was converted into precursor plasmid pDHFRI (Berkn et and 5harp+ Nuc, Ac1ds Res, +DHPR' cDNA Boel et al. (FBBS Lett, 219: 181-188.198 7) from the wild type DHFR cDNA. In summary, Plasmid pDHFRI was digested with BglI and BaIllI to generate DHFRc. A 1050 bp fragment containing the DNA was isolated. This Bgl II-BaIll 1 fragment was linearized by digestion with BasoHI, pEMBL 8 (Den te et al., Nuc, Ac1ds Res, 11: 1645-1655.1 983) by three-way ligation. Plasmids containing inserts in the correct orientation is called pDHFR/pEMBL8. Single chain pDHFR/pEMBL 8 DNA was prepared and Zoller and Sm1th (DNA, 3:47 9-488.1984) and mutagenic oligonucleotide ZC165 (5' GAG GCCAGGGTCGGT CTCCG 3’) subjected to constant in vitro mutagenesis. Mutagenesis using ZC165 of DHFR cDNA A mutation from Leu to Arg was introduced at position 22, and Simousen and Levi nson (danroc.

Natl, Acad, Sci, USA, 80: 2495-2499.19 This resulted in the DHPR' mutant cDNA described by (83). label Positive clones were identified by colony hybridization with ZC165. and the mutations were confirmed by dideoxy sequencing.

Replicated DNA was prepared from positive plaques and digested with Xho I [DHP A fragment containing the R' cDNA was isolated. Plasmid pDFIPR was transformed into Bgl [and Xho II pD) I digested with BatgHl and vector-containing sequences isolated The FRI fragment was ligated to the DHPR' cDNA fragment by two-way ligation. The plasmid containing FR' cDNA is pDHPJ? ’　I (first table).

Plasmid pD5CAT contains CAT-D) IFR' polycistronic transcription unit and was created from the ancestral plasmid pD5. Do as follows The chloramphenicol transacetylase (CAT) gene was transferred to pD5 B. Plasmid pD5CAT was created by inserting into the amH1 site. Plasmid pD5 was linearized by digestion with BamHI. CAT cDNA BamHI was obtained as an XhoU fragment and ligated to linearized ρD5 by two-way ligation. Correct The plasmid containing CAT cDNA in the opposite direction was named pD5CAT (see Figure 1).

As shown in Figure 1, a polycistronic transcription unit was created in pDSCAT. Ta. Plasmid pDHFR'I was digested with Fnu4HI and the attached end T at the end, DNA polymerase and four types of deoxyribonucleotide triphosph Smoothed by treatment with acid. Kinase treated linker blunted fragment, and this linker-added fragment was injected with BamHI and NcoI. It was digested by Gel a 0.62 kb fragment containing a part of DHPR' cDNA Isolated by electrophoresis and electroelution. Plasmid pDl (FR’I further Digested with Xba I and partially digested with Nco I to produce DHFR' 3'-end of cDNA, SV40 polyadenylation signal and 276 A fragment consisting of bp f7) I containing the pBR322 vector sequence was isolated. P Lasmid pD5CAT was digested with XbaI and partially digested with BamHI. The SV40 polyazonylation signal and the 276 bp pML vector sequence The taken XbaI-BanHI vector and the CAT gene of pD50AT were transferred to N co I-Xba I fragment and Ba1Ill I-Nco I fragment and ternary Connected by knot. The resulting plasmid was named pD5CAT-DHFR'. .

Figure 1 shows the 3' BamH1 site of the SV40 polyazonylesion signal. Plasmid pD5CAT-DHPR' was modified to change the C1a1 site to Illustrated. Plasmid pD5CAT-DHFR' to Xba11 and Sst Digested from I, DHFR' cDNA (7) part, SV40 and I77 A 0.9 kb fragment comprising the cation signal and the pML-1 vector sequence was isolated. This 0.9 kb fragment was linearized with Xba4-5stl and It was ligated to C3 by ligase treatment. The resulting plastic called pBoe1360a The Smid was digested with Ba1l I and DNA polymerase I (Klen o-fragment) and four types of deoxyribonucleotide triphosphates. Therefore, I filled in. The DNA was recircularized by ligation and pBoe13 The resulting plasmid containing a Claal site in place of the BamH1 site at 60a It was named pBoe1360b. Next, plasmid pBoe1360b was transformed into χba Digested with T and 5stl and isolated the 0.9 kb insert. Plasmi) 'pD5CAT-DHFR' was digested from Xba1 and 5stl, D)l FR', cDNA (7) 3'-partial SV40 polyazonylation sig A 0.9 kb fragment comprising the null and pML vector sequences was taken. pD5CA T-D) The XbaI-5stl vector-containing fragment from IFR' was converted into pBoe1 360b was ligated to the 0.9 kb Xba T-5stl fragment. The resulting product The lasmid was named pBoe1360 (Figure 1).

Stow (J, Virol, 37': 171-1'80.1981) Berkner and 5harp' (Nuc, Ac1ds Res', 11 : 600'3-6020.1983) 239 cells were infected with pBoe1360 and incomplete Ad5 viral DNA. Recombinant adenovirus vectors were generated by co-transfecting . The BglII site of mu9.4 of Ad5 was replaced with the Xba1 site, and A 100 mu fragment of ba19.4 was prepared. This fragment was converted into XbaI-digested pB oeL360 and by the calcium phosphate method using 10 μm of DNA. cells were transfected. Recombinant virus was recovered and Ad5. (C.A. It was named T-DHPR).

Flame ball GEMcGM Eco 1100'' human granulocyte macrophage colony The portion of the Ll-3 leader fused to the stimulatory factor coding sequence (hGM-CSF) was The cDNA to be coded was transferred to pDgGMU (Kaushansky et al., Bioch emistr, 26: 4861-4867, 1987) from hGM-C Cloned from mRNA isolated from COS cells transiently expressing 5F did. In summary, the 2.6 kb BstE II encoding hGM-C5F - EcoRI genome fragment BstE II - pD3 cut with EcoRI ( Example 1). Xaush the resulting plasmid pDgGMU ansky et al. 01-3105.1986). was affected.

Chirgwin et al. (Biochemistr., 18: 5294-5 299.1979) by substantially the same method as described by Using RNA as a poetic form, Gubler and Hoffman (Gene, 25: 263-269, 1983) 4 Even if the method described here is adapted A λgtll cDNA library was prepared.

By hybridization to a labeled hGM-C3F genomic probe Identified positive cDNA clones extend from the cap site through the coding region to the 3'-non- It was found that it contains the DNA and A sequences that fall into the translated region. hGM-CSF Co. A complete cDNA clone comprising a portion of Ll-3 fused to the code sequence was pGEM-1 (Pro Unique EcoR1 site of mega Bfotec, Madison, [) connected to. The resulting plasmid designated pGEMcGM, Eco, 1], oO hGM-C5F cD tandemly oriented in pGEM-1 vector It contained two copies of NA.

Example 5. TPC1a and d5 (11 polycistronic transcription units of TPCIJ) The Ll-3 leader sequence used in the intercistronic region of 8d5o ri, Ad2 major late promoter (MLP), Ad2 Ll-3 and its 5' splice signal associated with immunoglobulin 3' splice signal, DHFR cDNA S V40 polyazo=Lz-'-si57 signal and pUc1 Plasmid Ds/PIIC and plasmid pGE, comprising 3 vector sequences. Derived from McGMJco, 1100 (Example 4). As shown in Figure 2, Sumid pGEMcGM, Eco, 1100 was digested with Ba order and L1-3 lea A 0.8 kb fragment comprising the C3F cDNA fused to the vector sequence was isolated. . Oligonucleotide ZC582 (5' AAT TCCCGGG3') and Z C583 (GG 3' to 5' GTA CCCC) was treated with kinase and its Edited by Maniatis 1 plate A Lat 1 Sokyo Manu common in al+ Co1d Spring Harbor, NY+ 1982 Annealed using the conditions described in . Kinase treated and annealed The oligonucleotides form a BamHI-EcoRi adapter, which is ．． The 8 kb pGEMcGM, Eco, 1100 fragment was linked by ligase treatment. concluded. This ligation mixture was digested with EcoRl to contain the Ll-3 leader sequence. A 0.2 kb fragment consisting of

Plasmid pUc13 was linearized by EcoRI digestion and then prevented from recircularization. The cells were treated with calf intestinal phosphatase to stop them.

The 0.2 kb Ll-3 fragment was added to linearized pUc13 by ligase treatment. Connected. The resulting plasmid was named CSF Ll-3112 (1265). (Figure 2).

The 5' portion of the Ll-3 sequence was removed from pD'/PUC prepared as follows. It was. Plasmid pDs/PUC was obtained from Berkner and 5harp (supra, 1985) from the plasmid pDHFRnI. plus 5st between ^d5 ori and Ad2 MLP by modifying mid-pDHFRIII One unique Kpn site was placed in 11 sites.

Plasmid pD) IFRI[[ was linearized by 5stlI digestion and this line The shaped fragments are denatured by treatment with T, DNA polymerase and appropriate nucleotides. It was smoothed out. The linear fragment was ligated to a kinased Kpn I linker. excess The linker was removed by KpnI digestion, and the linker fragments were self-ligated. . The resulting plasmid was named pDHFRI[[/Sst4KpnI].

Plasmid pDHFRIII/Sst-+KpnI with EcoRI and Digest with Xba I and slice Ad50rl + Ad2 MLP% 5’. gnal, 3' splice signal, DHFR cDNA, SV40 polyazonyl - 2 comprising a John signal and approximately 300 bp of pBR322 vector sequence. , a 4 kb fragment was isolated. This 2.4kb fragment was injected into EcoRI and XbaI. This was then ligated to further digested and linearized pUc13. The resulting plasmid is pDs/P It was named t1C.

As shown in Figure 2, plasmid pD''/PUC was erased with EcoRI and Pstl. By converting Ad5 ori, Ad2 MLP and Ll-3 and 5′ A 1.1 kb fragment comprising the - and 3'-splice signals was isolated. this By digesting the 1.1 kb fragment with Hha I, Ll-3 and related 5 A 500 bp fragment comprising the '- and 3'-splice signals was isolated. Next Then, the cohesive ends were removed by treating the 500 bp fragment with T, DNA polymerase. Smoothed. This blunt-ended fragment was subjected to kinase treatment with Ba+sHI phosphorus. connected to the car. Excess linker was removed by digestion with BamHI; The fragment to which a linker was added was inserted into the BamH1 site of pUc13. Birth The resulting plasmid was named Ll-38am-Pst (#301) (Figure 2).

Figure 2 shows the construction of plasmid pTP (11323). Plasmid CSF By digesting Ll-3112 (#265) with Xba I and Nde I , 2.5 kb comprising the 3' portion of Ll-3 and pUc13s vector sequences. The fragment was isolated. Plasmid Ll-38am-Pst (#301) was added to BamHI 0,1 comprising the 5' portion of Ll-3 by digestion with A 7kb fragment was isolated. E. coli vector pUc13 with BamHI and NdeI A 200 bp vector fragment was isolated by digestion with . Ll-3BamHI -0.17kb Ll-3 fragment from Pst I (#301) was transferred to CSF L 2.5 kb fragment from l-31112 (#265) and 200 bp of pUc13 The fragments were joined by three-way ligation. The resulting plasmid was called pTP (1323). (Figure 2).

By digesting plasmid pTP (#323) with BaIIIH■ The Ll-3 fragment of kb is isolated. Plasmid pBoe1360 (C1a I ($ 221) and described in Example 2) as BarllH The plasmid was linearized by partial digestion with I. Linearized pBoe A 0.2 kb BamHI fragment of pip ($1323) was created by two-way concatenation of 1360. Connect to pieces. The ligation mixture was transformed into E. coli HBIOI. Plasmid DN A was isolated from transformants and analyzed by restriction enzyme digestion to determine the identity of the component fragments. proved the right direction. Insert in the correct direction between the cistrons between CAT and DHPR The plasmid containing the Ll-3 fragment was named pTP/C1a (!1431). (Figure 2).

using plasmid pTP/C1a, essentially as described in Example 3. Adenovirus vector Ad5 by transfecting 293 cells (TPCla) was produced.

l　D) Effects of cistron day-゛- on the level of IFH DHP with Ll-3 in the intercistronic region upstream of the DIlPR' coding sequence To establish increased transcription efficiency of R', Ad5 (CAT-DHFR) or Ad 5. (TPCla) DHPR protein in cells infected with recombinant virus The level of quality was measured.

293 cells that did not reach confluence were treated with Ad5 (CAT-DHFR) or were infected with Ad5 (TPCla) recombinant virus. ink infected cells cells were incubated and harvested when cells showed significant cytopathic effects. obtained The cells were pelleted by centrifugation, and the spent medium was discarded. phosphate buffer Remove excess medium by rinsing once with (PBS; Sigma, St. Louis, MO). was removed. Resuspend the cells in 0.25M Tris-HCI (pH 7,4). Ta. Lyse the cells by freezing and thawing the cell suspension and repeating this three times. I understand. Cell debris was removed by centrifugation and the 50I supernatant was collected in a 50m sample. Mix with pull buffer (Table 1) and electrophoresis on a 15% acrylamide gel. did.

Back - 1 side 361d0.5M Tris-HCI (pH 6,8) 16aj! Glycerol 16#! i! 20% SDS Mix all ingredients. Immediately before use, add 1004 β to the 900 Δ dye mixture. - Add mercaptoethanol.

Wesn°'A 30d IM Tris-HCI (pH 7,4) 20Id 0.25mM E DTA (pH 7,0) 5d 10% NP-40 37,5JI11! Tris+EDTA+ using 4M NaCl distilled water Dilute NP-40 and NaCl to a final volume of 12. 300d of this solution Add gelatin and run in microweb until gelatin dissolves into solution. Heat. Add the gelatin solution back to the remainder of the first solution and cool until cooled. Stir at 4°C.

The protein was also prepared by Towbin et al. (Proc, Natl, Acad, Sci, US A, 76:4350-4353.1979). and transferred to a nitrocellulose filter. nitrocellulose filter It was immersed in Western buffer A (Table 1) for 1 hour at room temperature. Remove the buffer and and anti-DHPR antibody diluted in Western buffer A for 1 hour at room temperature. Probed Luther. Remove the antibody solution and wash with Western buffer A 3. Excess antibody was removed from the filter by washing twice. bound by antibodies 125I-labeled protein diluted in Western buffer A. Visualization was achieved by incubation with A for 1 hour at room temperature. labeled Discard the protein A solution and wash three times with Western buffer A. Excess levels were removed by

X-rim the labeled filters for 4 hours at -80°C using a reinforced screen. exposed to film. According to this result, Ad 5 (TPCla ) jnpr protein produced by cells infected with the recombinant virus has high levels. However, it was infected by the Ad5 (CAT-DHFR) recombinant virus. It was not detected in the cells.

To confirm the production of polycistronic mRNA, BHK cells were incubated with p05CAT- Controls containing DHPR' or monocistronic DHPR expression units transfected with plasmid. produced by transfectants The obtained mRNA was added to a 32p-labeled antisense I) NA probe (Figure 3-B). hybridize and digest the mixture with 31 Uclairage to hybridize. The unsoyed probe was removed. Analysis of the product (Figure 3A) shows that pD5CAT-DHFR' directs the expression of nidistronic mRNA. (lanes 1 and 2, arrows), while monocistronic plasmids have a smaller mR He ordered the production of NA. The doublet observed in the distronic message is the first 3 by splicing in parts of the transcript as shown in Figure B. lane and The top band at 2 represents undigested probe DNA.

Polycistronic expression unit containing an intercistronic tripartite leader mRNA produced from cells transfected by Nor thern) plot (Thomas, Proc.

Analyzed by Natl, Acal, Sci, USA, Mutual, 5201.1980) did. °These results show that the prediction of distronic messages is mRNA of the expected size was produced.

f13. 71 factor IX cDNA of TP F9 C1a, CAT remains The tricistronic transcription unit comprising the gene and DHFR' cDNA is Constructed from Factor IX cDNA and pBoe136Q as shown in Figure 4. Manufactured. Plasmid pBoe1360 (Example 3) was partially digested with BamHI. The plasmid was linearized. As described in Maniatis et al. The cohesive ends were treated with bovine alkaline phosphatase in substantially the same manner as , which prevented self-concatenation. Kurach i and Davie (Proc, Natl. , Acal, Sci, UAS, 79: 6461-6464+1982' 1.4 kDNA of Factor ■ was prepared as described by It was isolated as a bBamH1 fragment. Next, this 1.4 kb BamHI fragment was It was subcloned into pUc13 to generate plasmid pF9pUc. P Digest lasmid pF9pUC with BamHT to generate 1.4kb Factor IX cD Isolate the NA fragment and insert it into linearized pBoe1360 by ligase treatment. Connected. The ligation mixture was transformed into E. coli strain 88101. plasmid DNA were prepared and screened by restriction enzyme digestion. In the correct direction and C Contains a Factor IX cDNA fragment inserted 5' of the AT cDNA sequence The plasmid was named pF9/C1a (Figure 4).

The tripartite present in plasmid pTp (#323) is extracted as follows. The leader sequence is the translation stop codon of Factor IX cDNA and CAT cDNA. It was inserted between the translation initiation codon of Plasmid pF9/C1a was incubated with BamHI and and Pstl to contain the 5'-coding sequence of Factor IX cDNA. A fragment (1.4 kb) consisting of Plasmid pTP (#323) (example 5) was linearized by partial digestion with BamHI and treated with phosphatase. Processed. Linearized pTP was prepared using PstI-BamHI adapter. Factor ■ Continuous to fragment.

Oligonucleotide ZC2029 (5' GAT CTCACCGTCTGCA 3') and ZC2030 (5' GACGGT GA 3') The adapter destroyed the Bam1 (I site but not the PstI site on the Factor ■ fragment). Saved the position. The resulting plasmid was named IX (TP) (1515).

Plasmid pP9/C1a’c Digested with AvaT and HindI[r, A 4.9 kb fragment containing the pML-1 sequence was then recovered.

Plasmid pF9/C1a was further digested with old ndlI and SspI, and MLP, 1,5k containing LL-3 and 5'-factor ■ sequences The b fragment was recovered. Next, the above two types of pF97C1a-derived fragments were added to plasmid I Ligated to Factor IX-LL-35spI-Aval fragment from X [TP) did. The resulting plasmid was named pTP/F9/C1a (Figure 4).

Plasmid pTP/F9/Cla and pF9/C1a are BHK cells were transfected and chloramphenicol acetyl trans The transient expression level of ferase was measured. pTP　 / F9 / CAT expression in C1a transformants was determined by pF97C1a transformation. It was about 20 times brighter than when it was replaced.

Flood p! L7 TP Bi2's Eisu A, made by Ueiri Toritsu The SV40 polyazonylesion signal is in the late oriental direction. From the vector plasmid pD3', which is the same as pH3 except in Plasmid pDX was created. Therefore, pD3' serves as the gene insertion site for Bat. It has an aH1 site.

To form poχ, the EcoRI site in pD3' was cleaved with EcoRI, S1 Incubation with nuclease followed by ligation with <Bcll linker The site was converted to the Bc11 site.

Plasmid DNA was prepared from colonies identified as positive and transformed A 1.9 kb Xho I-Pstl fragment containing the restriction site was placed on an agarose gel. Prepared by pneumophoresis. In the second modification, pD3 ( Example 1) with Kinase-treated EcoRI-Bcl I adapter (oligonuc Reotide ZC525: 5'-GGAATTCT-3'; and ZC526 :5'-GATCAGAATTCC-3'') to create an expression vector. An EcoR1 site was created as a location for insertion of the coding sequence into the vector.

Positive colonies were identified by restriction analysis and colonies identified as positive 2, 3 containing modified restriction sites using plasmid DNA prepared from The kb Xho I-Pst I fragment was isolated. pD3 and pD3' fragments T, incubated with DNA ligase and transformed into E. coli 1 (BIOI). , and positive colonies were identified by restriction analysis. Contains the desired expression vector. The plasmid containing this was named pDX (Fig. 5).

B. No. 1 Mutsukuri Ad5 ori, d2 major late promoter and promoter to SV40 enhancer-2 Lipartite leader, 5'- and 3'-splice signals, factor c Expression vector comprising DNA and SV40 polyazonylation signal , plasmid pDX.

pDll, and pFVII (565+2463)/pDX (ATCCNα40 205) Make a blank.

As shown in Figure 5, pFV If (565+2463) exists in pDX. Factor 1: Modify the cDNA to remove the 3'-untranslated region.

Plasmid pFV U (565-i-2463)/pDX with EcoRI Digest and isolate the 2.4 kb Factor ■ cDN^ fragment. EcoRI fragment By digesting with Mbo II, factor 1, 4 containing the coding sequence The EcoRI-MboII fragment of kb is isolated. Mbo II BamHI Kinase treatment of synthetic oligonucleotides designed to form adapters and anneal. The 1.4 kb fragment was kinased with the adapter and ligated with pUc13 linearized by digestion with EcoRI and BamHI. Ta. The resulting plasmid pUcF was digested with EcoRl and BaII) (I). ．． A 4 kb fragment (fragment a) was isolated.

Ad5 was obtained by digesting plasmid pDX with EcoRI and bar. ori, SV40 enhancer-1Ad2 major late promoter and tripal Tight leader, 5'- and 3'-splice signals and pBR322 vector A 4.Okb fragment comprising the target sequence was isolated. Plasmid pD11 (Example 2 ) was digested with Ban+HI and A 0.5 kb fragment (fragment C) comprising the signal and pML-1 vector sequence. ) was isolated. To create pFV II, fragments a, b, and C are joined in a ternary ligation. More connected.

C, 11Ad5 ori of Boosmid Dll-BIP, SV40 enhancer -1Ad2 major late promoter and tripartite leader, 5'- and 3' - Splice signal, BiP cDNA and SV40 polyazonylation Expression vectors comprising the signal were expressed as plasmids pD11 (Example 2), pBi P (pUc12) and pDX (Figure 5).

Immunoclone”)::11 Binding protein’l (B1P) cDNA pUc12 (Munro and Pelham, Cedan, 46: 291-3 00.1986) as a 2.4 kb EcoRI fragment in BiP (p It was named Uc12). BiP cDNA is present in pDll as follows. subcloned into the expression unit. BiP cDN^ pUc12 vector It was isolated as two fragments from the target sequence. BiP(pUc12) at 3amHI and K A 1.6 kb fragment was isolated by digestion with Kpn I and Eco Digested with RI and isolated a 0.86 kb fragment. Eco plasmid pDx Digested with RI and XbaI to generate SV40 polyazonylation signal and A 0.5 kb fragment comprising the pML-1 vector sequence was isolated.

By digesting plasmid pD11 with BamHI and XbaI, Ad5 orz-, SV40 enhancer-2Ad2 major late promoter and tripal Tight leader, 5'- and 3'-splice signals and pML~1 vector A 4,0 kb fragment comprising the -sequence was isolated.

Plasmid pD11-BIP was created by ligating the above four fragments.

This plasmid contains Ad5 ori, SV40 enhancer-1 Ad2 major late protein. lomotors and tripartite leaders, 5'- and 3'-splice signals , BiP cDNA, SV40 polyazonylation signal and pML- It includes one vector and one array.

D. Boosmid FVII-TP-BIP (7)” As shown in Figure 5 , Factor ■cDNA and BiP cDNA-containing distronic transcription unit Plasmid pFV II.

Constructed from pDII-BIP and pTP.

Plasmid pD11-RIP was digested with EcoRI and EcoRV to create BiPc A 0.5 kb fragment comprising the 5' portion of the DNA is isolated. 0.5kb fragment EcoRV-XbaI synthetic adapter and EcoRI and XbaI ligated to pUc13 linearized by digestion. Translate the resulting plasmid Nae I, which cleaves the BiP fragment at 5 bp 5' from the start codon. It becomes linear due to digestion. This linearized plasmid was synthesized into awl I library. Recircularization is achieved by ligation using a linker. The resulting plasmid was transformed into Bal1lI. pTP linearized by the digestion used and digested with BamHI (Example 5) A 0.2 kb BamHI fragment containing the tripartite leader obtained from Link. The resulting plasmid was partially digested with Bawl I and digested with EcoRV. By complete digestion, the tripartite leader and 5' BiP coding sequence A 0.7 kb fragment comprising .

The remaining fragments are obtained as follows. Plasmid pFV II was transformed with HindI [I and The Ad2 major late promoter and the Partite leader, 5'- and 3'-splice signals and factors■ A 2.1 kb fragment comprising the cDNA is isolated. Plasmid pD11-BIP SV40 enhancer-2 by isolating with old ndlI [and EcoRV Ad5 ori, pML-1 vector sequence, SV40 polyadenylation sequence 5.5 kb fragment comprising 3'-1.9 kb of Gnar and Bip cDNA isolate.

31! yr piece (0.5kb tripartite leader and 5'-Bip, 2, 1 kb (7) pFV II, 5.5 kMD pDll-BIP) in three rows Concatenate by original concatenation. The resulting plasmid was called pFVn-TP-BIP. to be called.

FIG.1 FIC;, 2 1 koji 10 + 1st drink 1mu 11 middle CI + □011 2 ePC near 1J530 no'tsu 32: Sashi international investigation report

Claims (15)

[Claims] 1. The following formula: P-C1-(HEL-Cn)m (In the formula, P is a transcription promoter; C is a DNA sequence encoding a protein; HEL is a high efficiency leader; n is a positive integer greater than 0; and m is an integer from 1 to 8) and which provides enhanced expression of at least one cistron. tronic transcription unit. 2. The leader (L) located downstream of the promoter (P) and upstream of C1 The polycistronic transcription unit of claim 1, further comprising. 3. The leader is SV40 leader, adenovirus first leader, adenovirus Illustribaltite leader, adenovirus L1-IX leader and Oval The policy of claim 2, wherein the policy is selected from the group consisting of Bumin leaders. Stronic transcription unit. 4. Polycistrone according to claim 2, wherein the leader is a viral leader. Sexual transcription unit. 5. The virus leader is adenovirus first leader, adenovirus avian Baltite leader, adenovirus L1-IX leader and SV40 leader 5. The polycistronic transcription of claim 4 selected from the group consisting of unit. 6. The port according to claim 1, wherein the high efficiency reader is a high efficiency virus reader. Lycistronic transcription unit. 7. Claim 1, wherein C1 and Cn are subunits of a multi-subunit protein. The polycistronic transcription unit described in . 8. The multi-subunit protein is Factor VIII, platelet-derived growth factor. , an immunoglobulin and a histocompatibility antigen. Sexual transcription unit. 9. C1 is protein C, factor VIII, factor IX, factor X and factor VIII. 2. The polycistronic transcription unit according to claim 1. 10. Cn is BiP, S. cerevisi ae) KEX2 gene product, flotein S and vonWi llebrand), which encodes a protein selected from the group consisting of 2. The polycistronic transcription unit according to claim 1. 11. C1 encodes protein C, and Cn encodes protein S and S ．． encodes a protein selected from the group consisting of the S. cerevisiae KEX2 gene product; The polycistronic transcription unit according to claim 1, wherein the polycistronic transcription unit comprises: 12. C1 codes for factor VIII and Cn is von Bill The polycistronic transcription unit according to claim 1, encoding a blunt factor. to. 13. The polycistronic transcription unit according to any one of claims 1 to 12 Cultured cells derived from multicellular organisms that have been introduced. 14. The cultured cell according to claim 13, wherein the cultured cell is a mammalian cell. 15. A method for enhancing protein expression in cultured cells derived from multicellular organisms, the method comprising: , polycistronic transcription unit according to any one of claims 1 to 12. into cultured host cells derived from microorganisms, and the cultured host cells are cultured in a suitable medium. A method comprising propagating.