HK1027360B - Production of recombinant human lactoferrin - Google Patents

Description

Production of recombinant human lactoferrin

This application is a divisional application with application number CN93105290.4, filed on 24/4/1993.

The present invention relates generally to the field of iron-binding glycoproteins. More particularly, it relates to the production of recombinant human lactoferrin.

Human Lactoferrin (LF) is a member of the transferrin family of monomeric glycoproteins that bind iron. It was originally found in milk and can be present in colostrum in amounts of up to 7 g/l. Thereafter, LF was detected in other external fluids, such as tears, saliva, and mucous secretions, which were also present in the secondary particles of the polymorphonuclear leukocytes.

LF is a glycoprotein with a molecular weight of 78 kilodaltons (KDa) and has a bilobal structure with a large degree of homology between the C-and N-termini, which is evident both at the amino acid sequence and at the three-dimensional structure level. Each lobe binds reversibly with high affinity to a ferric ion, which is accompanied by bicarbonate binding. Proposed biological functions of lactoferrin include: resisting microbial infection, enhancing intestinal absorption of iron by infants and young children, promoting cell growth, regulating myelogenesis, and regulating inflammatory response.

Filamentous fungi have been successfully used as hosts for the industrial production of extracellular glycoproteins. Some strains of industrial use secrete glycoproteins in grams. In addition, filamentous fungi are capable of correct post-translational modifications of eukaryotic proteins, and many strains have been licensed by the U.S. Food and Drug Administration (FDA). Furthermore, both large scale fermentation techniques and downstream processing techniques are available.

At present, there is no efficient and economical method for producing human LF. Therefore, with the progress of an efficient method for producing human lactoferrin, the application of lactoferrin to nutrition and medicine and further research work to elucidate its mechanism of action will solve the long-standing need in the art and describe related methods for producing lactoferrin.

In one embodiment, the present invention provides a recombinant plasmid comprising human lactoferrin cDNA. The plasmid of the present invention is suitable for expression in eukaryotic cells and contains regulatory factors necessary for expression of human lactoferrin cDNA in the eukaryotic cells.

In another embodiment, the invention provides a transformed eukaryotic cell containing a recombinant plasmid. The eukaryotic cell is selected from the group of filamentous fungi including Aspergillus. The plasmid contains a plasmid vector into which a polydeoxyribonucleotide fragment encoding human lactoferrin has been inserted.

In another embodiment of the present invention, a method of producing recombinant human lactoferrin is provided. The method comprises culturing a transformant of a eukaryotic cell, the transformed eukaryotic cell containing a recombinant plasmid. The plasmid contains a plasmid vector comprising a polydeoxyribonucleotide fragment encoding human lactoferrin. Culturing on a suitable nutrient medium until human lactoferrin is formed, and subsequently isolating human lactoferrin.

In another embodiment of the present invention, a recombinant expression vector is provided. The vector contains a transcription unit comprising the following elements: (1) one or more genetic elements having a regulatory role in gene expression; (2) cDNA encoding human lactoferrin; (3) suitable transcriptional and translational initiation and termination sequences; and (4) a genetic element for screening those Aspergillus spores transformed with the vector.

In another embodiment of the present invention, a method of producing biologically active recombinant lactoferrin is provided. The method comprises synthesizing a sequence comprising an alternative marker gene, a promoter, a transcription termination sequence and a linker sequence; cloning the sequence to form a plasmid; digesting the plasmid with restriction endonuclease; inserting a cDNA encoding lactoferrin into the restriction site; and transforming the eukaryotic nuclei with a plasmid capable of expressing lactoferrin cDNA.

So that the manner in which the above recited features, advantages and objects of the present invention are attained and can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to the embodiments thereof which are illustrated in the appended drawings. Which are part of the present specification. It is to be noted, however, that the appended drawings illustrate only preferred embodiments of the invention and are therefore not to be considered limiting of its scope. Other equally effective equivalent embodiments are also contemplated by this invention.

FIG. 1 is a schematic diagram of an Aspergillus oryzae (Aspergillus oryzae) expression vector (pAhlfg.).

FIG. 2 southern blot analysis of transformed Aspergillus oryzae (Aspergillus oryzae) strains.

FIG. 3 RNA analysis of A07 of the transformed strain versus the control.

FIG. 4 secretion of recombinant LF and purified silver stained SDS-acrylamide gel analysis.

Figure 5 characteristics of recombinant human LF.

FIG. 6 cDNA sequence encoding human LF.

For the purposes of this application, the term "family of transferrins" means a family of iron transfer proteins including serum transferrins, ovotransferrins and lactoferrin. These proteins are all structurally related.

For the purposes of the present application, the term "vector" means a plasmid vehicle capable of allowing the insertion, propagation and expression of lactoferrin cDNA.

For the purposes of the present application, the term "host" means any eukaryotic cell, as long as it allows the lactoferrin expression plasmid to integrate into its genome.

For the purposes of this application, the term "promoter" means a DNA regulatory sequence that controls transcription of lactoferrin cDNA.

For the purposes of this application, the term "polyclonal cassette" means a piece of DNA that contains multiple restriction enzyme sites, which later allow the insertion of different cDNAs.

For the purposes of this application, the term "transformation" means the uptake of a plasmid by the relevant eukaryotic cell.

For the purposes of this application, the term "iron binding capacity" means binding⁵⁹The ability of Fe. Intact lactoferrin. LF can bind two iron atoms per molecule.

For the purposes of this application, the term "bioactive" means the biological activity of lactoferrin as measured by its ability to bind iron. Lactoferrin functions as a transferrin and must bind iron for biological activity.

All documents cited in this specification are herein incorporated by reference in their entirety.

The following examples are presented for the purpose of illustrating various implementations of the invention and are not meant to be limiting (factors) of the invention in any way.

Example 1

Fungal strains and transformation

The pyr G mutant strain used for the study was derived from a. oryzae (a 0711488). The pyrG gene from A.oryzae was mutagenized with 4-nitroquinoline-1-oxidation. According to Osmani, et al, j.cell.biol.104: the transformation procedure of 1495-1504(1987) was slightly modified to transform Aspergillus. Mixing conidia (1 × 10)⁶/ml) was inoculated in 50ml of YG medium (0.5% yeast extract, 2% glucose) containing 5mM uracil and 10mM uridine. Growth was carried out at 32 ℃ for 14-16 hours until germination tubes were visible. The germinated conidia were harvested by centrifugation and resuspended in 40ml of lysis mix [ containing 0.4M (NH)₄)₂SO₄50mM potassium citrate (pH6.0), 0.5% yeast extract, 0.12gnovozyme, 0.1g of Driselase, 100. mu.l of beta-glucuronidase, 0.5% sucrose and 10mM MgSO₄]. Protoplasting at 32 ℃ and 150rpm for 2-3 hours. After protoplasting, it was necessary to filter with sterile miracloth (miracloth) to remove all undigested hyphae. Protoplasts were harvested by centrifugation and incubated with 10 ml of 0.4M (NH) at 4 deg.C₄)₂SO₄1% sucrose and 50mM potassium citrate (pH6.0) were washed twice and then resuspended in 1ml of 0.6M KCl; 50mM CaCl₂(ii) a 10mM Tris-HCl (pH7.5) and placed on ice. Transformation was performed immediately after preparation of protoplasts. Protoplasts (100. mu.l) were added 3. mu.g DNA and 50. mu.l of 40% polyethylene glycol (PEG)6000, 50mM CaCl₂0.6M KCl and 10mM Tris-HCl, (pH 7.5). After the sample was incubated on ice for 15 minutes, 1ml of PEG solution was added and incubation was continued at room temperature for 30 minutes. The mixture was divided into small portions, placed in 3ml of 0.7% semi-solid minimal medium, 0.4% ammonium sulfate was added, and the plates were poured onto solid medium plates containing the same ingredients but solidified with 2% agar. Subsequently, all plates were incubated at 32 ℃.

Example 2

Plasmid construction

A brief schematic of the expression plasmid is shown in FIG. 1. The complete cDNA encoding human LF was patched with Klenow fragment of DNA polymerase I and then subcloned into the plasmid pGEM4 digested with Acc I and filled in, resulting in plasmid pGEMhLFc. To remove the Signal sequence (Signal sequence) of LF and to generate a 5' end in frame with the alpha-amylase sequence, pGEMhLFc plasmid DNA was amplified by Polymerase Chain Reaction (PCR) to obtain a 252 base pair (bp) lactoferrin fragment containing the HindII/AccI end. The oligonucleotide primers used were as follows:

5' terminal oligonucleotide, as shown in SEQ ID No.1 (SEQ. ID No. 1):

\(CTGGGTCGACGTAGGAGAAGGAGTGTTCAGTGGTGC)

3' terminal oligonucleotide, as shown in seq id No.2 (seq id no 2):

(GCCGTAGACTTCCGCCGCTACAGG)。

the PCR amplified fragment was digested with Hind II and AccI and subcloned into Hind II/AccI digested pGEMhLFc, yielding pGEMhLF. The a-amylase fragment of 681bp with Asp 718/Pvu II end, encoding the promoter, signal peptide sequence and alanine residue at the start of the mature a-amylase II gene, was obtained by PCR amplification of a.oryzae genomic DNA. The oligonucleotide primers were as follows:

an oligonucleotide at the 5' end, as shown in seq id No.3 (seq. id No. 3):

(GAGGTACCGAATTCATGGTGTTTTG

-ATCATTTTAAATTTTTATAT)

3' terminal oligonucleotide, as shown in seq id No.4 (seq id No. 4):

(AGCAGCTGCAGCCAAAGCAGGTGCC

-GCGACCTGAAGGCCGTACAG)。

the amplified DNA was digested with Asp 718 and Pvu II and cloned into Asp 718/Hind II digested pGEMhLF. The constructed plasmid pGEMahLF was digested with EcoRI and the resulting 2.8Kb α -amylase-lactoferrin fragment was subcloned into the unique EcoRI site of pAL3, thus yielding pAhLF^*. The synthetic oligonucleotide is provided in pAhLF^*The last 5 codons of the carboxy-terminal of the deleted lactoferrin (nucleotides 2138-2153); the first 180bp of the untranslated sequence from the 3' end of the A.niger glucoamylase gene was also provided by an artificially synthesized oligonucleotide. The constructed plasmid, pAhL FG, was used to transform a mutant pyrG strain of Aspergillus oryzae (A.oryzae).

Referring to FIG. 1, Aspergillus oryzae (Aspergillus oryzae) expression plasmid pAhLFG contains 681bp 5' -flanking sequence of the A.oryzae AMY II gene sequence, which includes the signal peptide sequence and the first codon of mature α -amylase. The cDNA encoding mature human lactoferrin was subcloned in the same frame downstream of these sequences, so that recombinant proteins could be produced by adding starch to the culture medium. The 3' untranslated region of the glucoamylase from A.niger (Aspergillus niger) provides a transcription terminator and polyadenylation signal. The plasmid also contains the Neurospora crassa (Neurospora crassa) pyr4 selection marker as well as the ampicillin resistance gene.

The plasmid construct pAhLFG for expression of human lactoferrin, contains a 681 fragment encoding the promoter and secretion signal peptide of the a-amylase II gene (AMY II) of aspergillus oryzae. The signal sequence also contains an alanine (Ala) codon at the beginning of the mature α -amylase protein, thereby forming a signal peptide sequence cleavage site (Leu Ala Ala) that is recognized by the endogenous α -amylase peptidase. The human lactoferrin cDNA encoding the mature protein was subcloned into the same framework immediately downstream of the AMY II gene under the control of a highly efficient starch inducible promoter. To stabilize the transcribed human LF mRNA, a 180bp fragment encoding the 3' untranslated region of the glucoamylase gene of Aspergillus niger (Aspergillus miger) was ligated into the unique BamHI site of the multicloner cassette just downstream of the human LFcDNA, providing a transcription terminator and polyadenylation signal. The plasmid also contains the N.crassa pyr4 selection marker. pyr4 is complementary to the pyr G auxotrophic mutation of A.oryzae, and thus those spores transformed with the plasmid can be selected by growth in the absence of uridine.

Example 3

Manipulation of genomic DNA

Oryzae DNA, expressed as Rafmussen, et al, j.biol.chem., 265: 13767-. The DNA was digested with EcoRI, the fragment size fractionated on a 0.8% agarose gel and transferred to nitrocellulose. Prehybridization and hybridization of nitrocellulose filters for Southern blot analysis were performed in 6 XSSC, 0.1% SDS and 0.5% dry milk at 65 ℃ for 16 hours. The hybridization solution contained 1X 10⁷cpm³²P-labeled lactoferrin cDNA probe (2.1 kb). The filters were washed in 2 XSSC, 0.5% SDS at room temperature for 30 minutes, followed by two washes in 0.5 XSSC, 0.5% SDS at 68 ℃ for 30 minutes each. The filters were dried, exposed to light by autoradiography at-70 ℃ for two hours, and then developed.

Referring to FIG. 2, Southern blot analysis was performed using the transformed A.oryzae strain. Genomic DNA from each transformant and A07 as a control was hybridized with a radiolabeled hL F cDNA probe (2.1 kb). The arrow indicates the radiolabeled fragment (2.8 kb). This fragment was generated by digesting the expression plasmid with EcoRI. Present in all transformants (#1-9) but absent in the control, non-transformed A07. The molecular weights of the HindIII digested fragments of phage lambda are listed on the left.

Example 4

Northern blot analysis

RNA was isolated from 200mg of freeze-dried mycelia using commercially available RHAZOlB (Biotech Laboratories, INC, Houston, TX) according to the manufacturer's instructions. Mu.g of total RNA was electrophoresed in 0.8% agarose gel containing 2.2M formaldehyde, and then the RNA was transferred to nitrocellulose and hybridized with 2.1kb lactoferrin cDNA or 1.8kb genomic alpha-amylase fragment corresponding to the coding region of the alpha-amylase II gene. The probe is used by nick translation³²p-labelled (specific activity 2X 10)⁸cpm/. mu.g). 2X 10 for hybridization⁶cpm probe/ml in 2 XSSC, 0.5% dry milk, 65 ℃.

The washing was performed in the same manner as that previously used for Southern blot analysis. The filters were dried, exposed to light at-70 ℃ for 2 hours, and then autoradiographed. RNA dot blotting was performed using a nitrocellulose membrane and a multi-purpose dot blotting system (manifold dot blot system m). Hybridization and washing conditions were the same as described in the souhetem blot analysis above. Radioactivity was counted using a beta particle blot analyzer (beta blot analyzer).

The production of recombinant lactoferrin has been described in its preferred embodiment. However, recombinant products may also be obtained from many other sources, for example using fungal sources such as Saccharomyces acidovorus or Pichia pastoris, or insect cells such as SF 9.

Referring to FIG. 3, northern blot analysis of transformants against control A07 is presented. FIG. 3A, Northern blot analysis of hybridization of RNA (20. mu.g) of control A07 and transformant #1 to radiolabeled human LFcDNA. Human LF mRNA (2.3kb) was detectable in transformant #1, but not in control untransformed A07. The positions of 28s and 18srRNA are listed on the left. FIG. 3B, point blotting of RNA (5 and 10. mu.g) of transformant #1 with control A07, using a radiolabeled alpha-amylase genomic DNA probe. FIG. 3C, dot blot of control A07 on RNA from transformant #1 (5. mu.g and 10. mu.g) using the indicated radiolabeled human LFcDNA probe.

Northern blot analysis was performed to determine if lactoferrin mRNA was transcribed correctly and efficiently in a. orzyae under the control of the regulatory factors of our expression plasmids. Untransformed spores (1X 10) from transformant #1 and control⁶/ml) was inoculated on a fungal culture medium containing 1.5% glucose as carbon source. The cells were cultured in a 30 ℃ small flask for 48 hours. The cultures were washed and inoculated into fungal media containing 3% starch to induce transcription of human LF mRNA. After 24 hours, cells were harvested and RNA was isolated. Total RNA (20. mu.g) was fractionated on a 1.0% agarose gel containing 2.2M formaldehyde and blotted on nitrocellulose.

Human LFmRNA was detected using a 32P-labeled human LF cDNA (2.0kb) probe. Hybridization with the radiolabeled human LFcDNA probe detected a specific radiolabeled band corresponding to the correct location of the LF mRNA (2.3kb) of the transformants, but not in the control untransformed strain (FIG. 3A). Quantitative analysis of the mRNA by dot analysis indicated that the control a07 was expressed at substantially the same level as the endogenous α -amylase mRNA of transformant #1 (fig. 3B). In addition, approximately equal expression levels of α -amylase and human LF mRNA were seen in transformant #1 (fig. 3B and 3C).

Example 5

Purification of recombinant human LF

Purification of LF from growth medium CM Sephadex C50 was used, essentially as per St owell, et al, biochem j., 276: 349-59 (1991). The column was pre-equilibrated with 500ml0.025M Tris HCl, pH7.50, 1M NaCl. The pH of the medium was adjusted to pH7.4 prior to the upper column (pre-equilibrated). The column was washed with 500ml of equilibration buffer and then linearly increased from 0.1 to 1.1M NaCl with a salt gradient. The fractionated washings (total 7ml) were analyzed for lactoferrin content and purity by SDS-polyacrylamide gel electrophoresis (SDS/PAGE) and silver staining. The fraction containing LF was dialyzed against 0.025M Tris HCl, pH7.5/0.1M NaCl and freeze-dried.

Example 6

Quantification of human LF

Quantification of recombinant lactoferrin was analyzed by ELISA, essentially as described by Vilja et al, j.immunol.methods, 76: 73-83 (1985). Using a non-competitive avidin-biotin assay, the sensitivity was up to 5ng lactoferrin. Human LF (from Sigma) isolated from milk was used as a standard. Biotinylated human lactoferrin Ig is obtained from Jackson Immunoresearch laboratories, West Grove, PA.

Example 7

N-terminal sequencing

Mu.g of purified recombinant human LF was separated on SDS-polyacrylamide gel and transferred to Problott, a polyvinylidene fluoride type membrane (product of Applied Biosystems), according to the manufacturer's instructions (Applied Biosystems). Human LF was detected with coomassie brilliant blue staining and then destained. The LF band of human body was cut, washed thoroughly with distilled water, and air-dried. The first ten amino acid sequences at the N-terminus of human LF were determined by automated Edman degradation using an Applied biosystems ms pulse-liquid sequencer (Model 477A).

Referring to fig. 4, fig. 4A shows the results of silver-stained SDS-polyacrylamide gel analysis of recombinant human LF secretion and purification. Lane 1 milk containing human LF as standard (500 ng). Lanes 2 and 3 contained growth medium samples (40 μ g) from induced control A07 and transformant #1, respectively. Lanes 4-8 contained 100. mu.l each of the elution fractions (# 25, 30, 35, 40 and 45, respectively) collected from the growth medium of transformant #1 when recombinant LF was purified on CM-Sephade x. The position of the molecular weight markers (BioRad laboratories R ichmond, CA) is shown on the left. Molecular weight is measured in kilodaltons. FIG. 4B shows the results of Western immunoblot analysis of the same samples as in FIG. 4A, using specific polyclonal antibodies against human LF, which antibodies are useful¹²⁵I labeled protein A. FIG. 4C shows N-terminal ammonia of #6 recombinant human LFAn amino acid sequence. Recombinant human LF was sequenced 10 residues from the N-terminus and confirmed to be identical to that of milk human LF except for the additional alanine. This alanine is present in our construction and provides a cleavage site for the alpha-amylase signal peptide sequence.

Example 8

Deglycosylation

Deglycosylation was performed with N-glycosidase F (Boehringer Mannheim). A. oryzae growth medium containing 0.5 μ g lactoferrin was denatured in the presence of 0.01% SDS at 100 ℃ for 3 minutes. Standard LF obtained from human milk was treated the same. The sample was then placed on ice for 5 minutes. The N-glycosidase F reaction was carried out under the following conditions: 0.4M sodium phosphate, (ph 6.8); 0.08% Triton; 0.1% beta-mercapto2 alcohol and 1 unit of enzyme, incubated at 37 ℃ for 16 hours. PAGE and Western blot analysis was performed with IgG specific for human LF to assay the digested samples for an increase in mobility.

See fig. 5, properties of recombinant human LF. Panel a shows deglycosylation of LF. Using specific polyclonal antibodies against human LF, the antibodies can be used¹²⁵I-protein A was detected and Western blot analysis of glycosylated and deglycosylated lactoferrin was performed. The first panel is real milk human LF (500ng), including (+) without treatment (-) and with N-glycosidase F (+). The second panel shows purified recombinant human LF (500ng) including (+) without N-glycosidase F treatment (-) and (+) with N-glycosidase F treatment. The molecular weight of glycosylated human LF is indicated by the arrow. Panel B shows functional testing of recombinant LF for its ability to bind iron. Panel A and B show identical real milk human LF and purified human LF samples, respectively⁵⁹Fe filter binding assay was performed at the concentrations indicated in the figure. The first lane of both panels contains BSA (5. mu.g) as a negative control.

Lactoferrin contains two N-acetyl lactosamine-type glycans linked by N-glycosidic bonds. To determine whether recombinant LF was correctly glycosylated, recombinant lactoferrin was treated with N-glycosidase F, separated on SDS-polyacrylamide electrophoresis, transferred to nitrocellulose membrane, and detected with IgG specific for human LF as a probe (fig. 5A). Hydrolysis of N-glycosidase F at the sugar amine linkage produces a smaller molecular weight carbohydrate-free polypeptide. Comparing recombinant LF with LF purified from human milk, it can be seen that both proteins co-migrate after digestion with N-glycosidase F, which means that the recombinant protein has a glycosylation pattern similar to that of native LF.

Lactoferrin has a bilobal structure with each lobe having a strong and reversible binding of one 3Fe³+ ionic capacity. The iron-binding properties of lactoferrin are critical for its function. To test whether recombinant human LF expressed and secreted in a. oryzae has similar ability to bind iron as authentic LF, an assay was developed⁵⁹Fe microfiltration membranes are combined with analytical techniques. Purified human LF isolated from the growth medium of transformant #1 was dialyzed against 0.1M citric acid (ph2.0) to yield apop-human LF. Natural LF from human milk was treated the same. Adding in excess⁵⁹Fe (0.2mCi) was added to both samples (samples dissolved in the same volume of 1M bicarbonate) followed by incubation at 37 ℃ for 30 minutes. The sample was then transferred to a nitrocellulose membrane and washed several times with bicarbonate solution. The filters were autoradiographed and then the iron binding was quantified using a beta particle blot analyzer. As shown in fig. 5B, the levels of bound iron were similar for recombinant LF and native LF at all concentrations tested. The results show that recombinant human LF is not different from natural human LF in its ability to bind iron.

Referring to FIG. 6, showing the complete cDNA sequence of human lactoferrin, the cDNA encoding lactoferrin was used to construct plasmids, transform eukaryotic cells and produce lactoferrin.

The Aspergillus strains used in the present invention are auxotrophic mutants that contain a defective pyr4 gene and are therefore unable to synthesize orotidine 5' -phosphate (OMP) decarboxylase. This enzyme is essential for the synthesis of uridine. Thus, the deficient strain cannot grow on a medium lacking uridine. The plasmid contains an alternative marker gene, the sequence encoding the OMP decarboxylase gene. Thus, the Aspergillus uptake of this plasmid allows selection for growth on media lacking uridine. This plasmid was used to transform Aspergillus to allow growth on a medium lacking uridine.

In one embodiment of the invention, biologically active recombinant lactoferrin is produced. The method comprises the following steps: synthesizing a sequence comprising an alternative marker gene, a promoter, a transcription termination sequence and a linker sequence; subsequently cloning the sequence to form a plasmid, and digesting the plasmid by using restriction endoribozymes; inserting a cDNA encoding lactoferrin into the restriction site; the eukaryotic cells were then transformed with a plasmid expressing lactoferrin cDNA.

The marker gene used in the method of the present invention may be any gene as long as it can isolate cells transformed with the LFcDNA plasmid. Preferably, the selectable marker gene is selected from pyr4, pyrG, argB, trpC and andS.

The promoter used in the present invention may be any one that allows regulation of transcription of LFcDNA, and preferably, the promoter is selected from the group consisting of: a yeast dehydrogenase, argB, an alpha-amylase and a glucoamylase.

The transcription termination sequence used in the present invention may be any one as long as it can stabilize LFmRNA. Preferably, the transcription termination sequence is derived from alpha-amylase, glucoamylase, alcohol dehydrogenase or ben A.

The linker sequence used in the method of the present invention may be any linker sequence as long as it contains a translation initiation codon, a secretion signal and a restriction enzyme site. Preferably, the linker sequence is derived from alpha-amylase, glucoamylase or lactoferrin.

The eukaryotic cell used in the present invention may be any eukaryotic cell as long as it can be integrated with a plasmid containing LF cDNA and express the LF cDNA. Preferably, the eukaryotic cell is a fungal cell or an insect cell. Insect cells such as SF9 can be used in the methods of the invention. More preferably, the fungal cell is a yeast cell. Most preferably, the eukaryotic cell used in the present invention is an aspergillus strain, such as: aspergillus oryzae (a. oryzae), aspergillus niger (a. niger), aspergillus nidulans (a. nidulans) and aspergillus awamori (a. awamori).

From the foregoing, it will be seen that this invention and the embodiments thereof disclosed herein are one well adapted to attain the ends and objects set forth herein. Certain changes can be made in the method and apparatus without departing from the spirit of the invention and without departing from the scope of the invention. It should be appreciated that these variations are possible. Further, it should be appreciated that each element or step in any claim herein is understood to encompass all equivalent elements or steps which perform substantially the same result in substantially the same or equivalent manner. The principles of the present invention, whether applied in any form, are intended to be applied more broadly. The present invention, therefore, is well adapted to carry out the objects and attain the ends and advantages mentioned, as well as those that are inherent therein.

Claims (5)

1. A method of producing lactoferrin comprising culturing transformed aspergillus fungal cells containing a recombinant plasmid on a suitable culture medium until lactoferrin is formed, said plasmid containing a plasmid vector carrying polydeoxyribonucleotides encoding lactoferrin; then separating the human lactoferrin from the culture medium.

2. A recombinant expression vector comprising a transcription unit consisting of the sequence set forth in seq id no: (1) one or more genetic elements that are regulatory in the expression of the gene in the fungal cell of the genus Aspergillus; (2) cDNA encoding human lactoferrin; and (3) suitable transcription and translation initiation and termination sequences.

3. The vector of claim 2, wherein said genetic element is a promoter.

4. The vector of claim 3, wherein said promoter is selected from the group consisting of: alcohol dehydrogenase, argB, alpha-amylase, glucoamylase and benA genes.

5. The vector of claim 2, wherein said transcription termination sequence is selected from the group consisting of alpha-amylase, glucoamylase, alcohol dehydrogenase and benA genes.