DE20207301U1 - Expression system for synthetic genes in microalgae - Google Patents

Description

Utility model: Expression system for synthetic genes in Mi

Registration of a utility model

Title of the invention: Expression system for synthetic genes in microalgae. This consists of a combination of gene sequences that lead to the export of proteins of any size, which are encoded by synthetic genes, from the cytosol of microalgae into the surrounding medium.

Short description

The functionally important gene sequence of the export system for synthetic genes in microalgae consists of a combination of any promoter, e.g. the HSP-r£cS2-Inl promoter, an export sequence (= leader e.g. of arylsulfatase = ARS or chlamyopsin 3 = COP3) from Chlamydomonas reinhardtü, a facultative synthetic N-terminal epitope (e.g. codes for S-tag and/or a protease recognition sequence e.g. factor Xa cleavage site), a synthetic structural gene, a facultative synthetic C-terminal epitope (e.g. codes for HSV-tag, protease recognition site etc.) and a 3' UTR e.g. of the native rZ>cS2 gene from Chlamydomonas reinhardtü.

Description

A wide variety of recombinant proteins are required in diagnostic and therapeutic medicine. Several expression systems have been developed for this purpose, but they all have their advantages and disadvantages in terms of the amount of protein produced and costs, etc. The demand for recombinant protein is forecast to continue to rise in the coming years. To meet this increasing demand, novel, safe and inexpensive production systems are needed.
To date, recombinant proteins for medical use have been obtained primarily from microorganisms (bacteria or yeast) or from animal cell cultures. One problem with the bacterial system is the lack of important secondary modifications that are necessary for the functionality of the proteins. The synthesized proteins are not further processed and are therefore often non-functional or they lead to an increased triggering of immune responses. Expression in mammalian cell culture is very costly and produces only small amounts of the desired protein. However, this has the

Utility model: Expression system for synthetic genes in microfluids

correct secondary modifications. Recently, more and more plant systems have been used for protein synthesis. Although proteins from plant cells have a different glycosylation pattern than proteins from animal cells ¹ , physiologically active and immunoneutral proteins (e.g. antibodies) can already be obtained from transgenic plants (e.g. tobacco). A major disadvantage of expression in plants is the risk of the possible spread of the transgenic properties (= gene transfer) to other plants in the area. In addition, time-consuming and costly purification processes are necessary to obtain the recombinant proteins (enzymatic cell wall degradation, cell disruption and multi-stage chromatography processes). Yields of up to 10% TSP (total soluble protein) have been described in isolated cases in the literature, with the average yields achieved being less than 0.2%. ²
This patent describes a combination of nucleic acid elements that is used to express synthetic genes in the microalgae Chlamydomonas reinhardtii , modify the gene products (proteins) and release them into the medium. A major advantage of this system is that the green algae can be cultivated in a closed system and there is therefore no risk of the transgenic elements spreading to other organisms. The key advantage of this system is the simplified purification of the expressed proteins. Since these are exported directly into the surrounding medium by the cell wall-less strains used, regardless of their size, the labor-intensive and costly steps of enzymatic or mechanical disruption of the cells are eliminated.

Example:

The synthetic gene encoding the luciferase from Renilla reniformis and adapted to the preferred codon usage of Chlamydomonas reinhardtii was cloned between the combination of the nucleic acid elements HSP-rbcS2-Inl promoter, export sequence (= leader) of the arylsulfatase from Chlamydomonas reinhardtii, N-terminal epitope (encoding S-tag and a factor Xa protease cleavage site), C-terminal epitope (HSV-tag) and 3' UTR of the native rbcS2 gene from Chlamydomonas reinhardtii in the vector pBluescript II KS" (from Stratagene). The cell wall-less C. reinhardtii strain cwl5 arg" was co-transformed with this vector and the plasmid pArg7.8 from Debuchy et al; (1989) ³ . The transformants were tested for complementation of the arginine auxotrophy of

'Bakker et al.; PNAS 98(5); 2899-2904; 2001

² Daniel! et al.; Trends in Plant Science 6(5) ; 219-226 ; 2001

³ Debuchy et al.; The EMBO Journal Vol. 8-10; pages 2803-2809; 1989

Utility model: Expression system for synthetic genes in microorganisms 1*1 I * * * *.*

Of the resulting Arg ⁺ transformants, 5-10% showed significant luciferase activity.

Separation of the cells from the medium revealed that almost all of the enzymatic activity of luciferase was in the extracellular medium, i.e. the active protein (42 kD) was transported out of the cells with high efficiency (see also Figure 5).

State of the art

1. There are currently various methods to transform genes in various micro- and macroalgae and to integrate them stably into the genome ⁴ ' ⁵ - ⁶ . Targeted integration into the nuclear genome ⁵ as well as into the genome of organelles ⁶ (chloroplasts and mitochondria) is possible.

2. Since the preferred codon usage in genes of green algae and plants in general differs from animal and human genes, which could be a reason for low expression rates, codon-adapted genes are often used to improve the expression rate. The de novo synthesis of nucleic acid polymers ⁷ has been developed to such an extent that it is offered routinely by various companies.

3. So far, as an example of the expression of recombinant proteins in green algae, the transient expression and secretion of hGH (human growth hormone, 17.5 kD) in the green alga Chlorella has been described in the literature ⁸ .

4. In normal cell wall-containing strains, proteins larger than about 30kD are not freely released into the

Medium secreted, but remain bound in the periplasmic region (in the area of the cell wall), eg carbonic anhydrase (35 kD), arylsulfatase (70 kD, dimerization), alkaline phosphatases (190 kD). These enzymes can be released into the medium through enzymatic degradation of the cell wall by gamete autolysin. Cell wall-defective strains (cwl5) secrete these proteins directly into the medium ⁹ , ¹⁰ , ¹¹ .

Apt et al.; MGG 252; 572-579; 1996

Kindle et al; J Cell Biology 109(6); 2589-2601; 1989

Blowers et al.; Plant Cell 1; 123-132; 1989

Fuhrmann et al.; Plant Journal 19(3); 353-361; 1999

Hawkins &Nakamura; Curr. Microbiol. 38 ; 335-341 ; 1999
⁹ Qui5eletal.;PlantPhysiol. 111; 839-848; 1996

Toguri, et al.; Eur J Biochem 158; 443-450; 1986

de Hostos et al.; J Cell Biol 106; 29-37; 1988

Utility model: Expression system for synthetic genes in microalgae I '' II * * II .*

Going beyond this, the invention described here generated stably transformed cell wall-less algae that secrete the recombinant protein quantitatively into the surrounding medium. The synthetic nucleic acid was integrated into the nuclear genome, thereby creating an inherited property of the stable transgenic line that results in the protein being permanently produced and exported.

Use pattern: Expression system for synthetic genes in microalgae*

Appendix: Illustrations

Figure 1 Figure 2

Schematic structure of a DNA used for the expression of foreign genes or gene fragments in algae.

Example sequence with synthetic structural gene encoding the luciferase of Renilla reniformis

Figure 3

Example sequence broken down into individual components from Figure

Figure 4

Gene and amino acid sequence in the example from Figure

Figure 5

Export efficiency of the overall construct from the nucleic acid elements described in Figure

Claims (13)

1. Nucleic acid polymers which represent a transcription unit, the nucleic acid consisting of at least the elements A, B and D ( Fig. 1), where A acts as a promoter, B encodes the export sequence and D represents the structural gene or gene fragment which encodes a protein which is to be synthesized and exported according to the invention. 2. Partially synthetic nucleic acid polymers which represent a transcription unit, the nucleic acid consisting of at least the elements A, B and D ( Fig. 1), where A acts as a promoter, B encodes the export sequence and D represents the structural gene or gene fragment. 3. Nucleic acids according to claim 1, wherein the nucleic acid polymer additionally contains one or more of the elements C, E and/or F ( Fig. 1), wherein C encodes an N-terminal epitope and E encodes a C-terminal epitope, which serve for protein detection and/or protein purification, and F represents a 3' untranslated region (UTR). 4. Nucleic acids according to claim 1, wherein B represents the export sequences of arylsulfatase = ARS or chlamyopsin 3 = COP3 from Chlamydomonas reinhardtii ( Fig. 3). 5. Nucleic acids according to claim 1, wherein A represents the HSP-RBCS2-In1 promoter according to Ref. ¹⁰ ( Fig. 3). 6. Nucleic acids according to claim 2, wherein O represents a synthetic nucleic acid polymer, preferably the S-tag [Novageil] and/or a protease recognition sequence, preferably for factor Xa ( Fig. 3). 7. Nucleic acids according to claim 5, wherein E represents a synthetic nucleic acid polymer which preferably encodes the HSV tag [Novagen] ( Fig. 3). 8. Nucleic acids according to claim 5, wherein F represents the 3' UTR of the RBCS2 gene from Chlamydomonas reinhardtii ( Fig. 3). 9. Nucleic acids according to claims 1 to 7, wherein individual elements A, B, C, E or F are mutated, but still have at least 80% homology to the original elements described under 1 to 8. 10. Vectors containing nucleic acids according to claims 1 to 9. 11. Microorganisms containing DNA or vectors according to claims 1 to 11. 12. Proteins encoded by nucleic acids according to claims 1 to 11. 13. Proteins produced and secreted by microorganisms according to claim 11.