A METHOD FOR RECOMBINANT PROTEIN PRODUCTION

Abstract

A method including the process steps of insertion of a gene to be expressed into the plasmid carrying AOX1 promoter (i), cloning of plasmid carrying gene (ii), transfer of recombinant plasmid carrying AOX1 promoter and gene to expression host (iii) and providing both induction of AOX1 promoter and expression of associated heterologous gene, using a nitric oxide donor (iv). The method enables expression of various genes of various microorganisms, plants, animals and human, and thus extracellular and/or intracellular production of various recombinant proteins.

Claims

1. A method for extracellular and intracellular recombinant protein production comprising the process steps of: i. insertion of a gene to be expressed into a plasmid carrying AOX1 promoter; ii. cloning of plasmid carrying gene; iii. transfer of recombinant plasmid carrying AOX1 promoter and gene to an expression host; and iv. providing both induction of AOX1 promoter and expression of associated heterologous gene, using a nitric oxide donor.

2. The method according to claim 1, wherein the gene mentioned in process step no (i) is a nucleotide sequence of protein intended to be produced recombinantly and is obtained from a living being selected from a group comprising human, plant, animal and microorganisms.

3. The method according to claim 2, wherein the gene mentioned in process step (i) being a prepromelittin protein gene of honey bee.

4. The method according to claim 2, wherein the gene mentioned in process step (i) being a PINX1 protein gene.

5. The method according to claim 1, wherein the plasmid mentioned in process step (i) being selected from a group comprising pPICZα (A, B and C) plasmid, pPICZ (A, B and C) plasmid and other P. pastoris plasmids carrying AOX promoter.

6. The method according to claim 1, wherein the bacteria cells are used for cloning of heterologous gene inserted into plasmid in process step no(ii).

7. The method according to claim 6, wherein said bacteria cells are E. coli cells.

8. The method according to claim 7, wherein said E. coli cells are E. coli TOP10 strain cells.

9. The method according to claim 1, wherein the expression host mentioned in process step no (iii) being selected from a group comprising yeasts (Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia hpolytica and Kluyveromyces lactis), bacteria (Esherichia coli, Bacillus subtilis, and Lactococcus lactis) and plants (Arabidopsis sp. and Nicotiana tabacum).

10. The method according to claim 9, wherein the expression host mentioned in process step no (iii) is the recombinant P. pastoris cells that carry the protein gene transferred to plasmid and perform production of this protein thereof extracellularly.

11. The method according to claim 1, wherein NO donor used as inducer in process step no (vi) being selected from a group comprising sodium nitroprusside, glyceryl trinitrate, isoamyl nitrite, isosorbide dinitrate, isosorbide mononitrate, s-nitrosoglutathione, s-nitroso-n-acetylcysteine, s-nitroso-N-acetylpenicillamine, diethylamine NONOate and spermine NONOate.

12. The method according to claim 11, wherein NO donor is sodium nitroprusside.

13. The method according to claim 11, wherein at least a carbon source selected from a group comprising sorbitol, mannitol, trehalose and glycerol being used additionally in the process step no (vi).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 is efficiency analysis graphic of heterologous gene expression levels giving comparison of method of invention to control groups of different applications.

DESCRIPTION OF REFERENCES

[0030] A. Growth culture containing methanol in 0.5% in volume

[0031] B. Growth culture containing glucose in 1% and methanol in 0.5% in volume

[0032] C. Growth culture containing glucose in 1% and 800 nM sodium nitroprusside

[0033] D. Growth culture containing glycerol in 1% and methanol in 0.5% in volume

[0034] E. Growth culture containing glycerol in 1% and 800 nM sodium nitroprusside

[0035] F. Growth culture containing sorbitol in 1% and methanol in 0.5% in volume

[0036] G. Growth culture containing sorbitol in 1% and 800 nM sodium nitropusside

DETAILED DESCRIPTION OF THE INVENTION

[0037] In this detailed description, a method for recombinant protein production being subject of this invention and the preferred applications have been disclosed for the purpose of better understanding of the subject and described in a manner not causing any restrictive effect.

[0038] Invention is a method for extracellular and intracellular recombinant protein production comprising process steps of [0039] i. Insertion of gene to be expressed into the plasmid carrying AOX1 promoter [0040] ii. cloning of plasmid carrying gene [0041] iii. transfer of recombinant plasmid carrying AOX1 promoter and gene to expression host [0042] iv. providing both induction of AOX1 promoter and expression of associated heterologous gene, using a nitric oxide donor.

[0043] ‘gene” mentioned in process step number (i) is a nucleotide sequence of protein intended to be produced recombinantly and is obtained from a living being selected from a group comprising human, plant, animal and microorganisms.

[0044] In an application of the invention said gene is prepromelittin protein gene of honeybee (Apis mellifera). In another application of the invention, said gene is human PINX1 (hPINX1) protein gene.

[0045] According to a preferred application of the invention said plasmid is selected from a group comprising Pichia pastoris plasmids carrying AOX promoters such as pPICZα (A, B and C) and pPICZ (A, B and C).

[0046] In an application of the invention, in processes step no (ii) bacteria cells are used for cloning of heterologous gene inserted into plasmid. In a preferred application of the invention said bacterial cells are E. coli cells, most preferably E. coli TOP10 strain.

[0047] In another application of the invention, said expression host in process step no (iii) is selected from a group comprising yeasts (Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Kluyveromyces lactis), bacteria (Escherichia coli, Bacillus subtilis and Lactococcus lactis) and plants (Arabidopsis sp. and Nicotiana tabacum).

[0048] In a preferred application of the invention the expression host mentioned in process step no (iii) is the recombinant P. pastoris cells that carry the protein gene transferred to the plasmid in its genome and perform production of this protein thereof extracellularly.

[0049] In a preferred application of the invention, nitric oxide donor mentioned in process step no (iv) is used as inducer.

[0050] Nitric oxide (NO) is a water and fat soluble gas molecule produced endogenously in bacteria, yeast, plants and humans. The molecule has different physiological functions in said organisms. For instance, it is reported that in higher plants endogenous NO plays a significant role in plant growth and development, seed germination, primary and lateral root growth, flowering, fruit ripening, senescence, respiration and stomatal closure, and adaptive responses against biotic-abiotic stresses. NO produced as endogenous in human body functions as a signal molecule and plays role in the regulation of immunological responses, blood flow, secretion, oxygen perception and respiratory energy reproduction.

[0051] Due to mentioned beneficial properties of NO, the application of exogenous NO to biological systems for biotechnological and medicinal purposes is seen as an important approach. In this line, NO donors providing exogenous NO are applied to biological systems. Released exogenous NO mimics response of endogenous NO or completes its deficiency.

[0052] In a preferred application of the invention, NO donor is selected from a group comprising sodium nitroprusside, glyceryl trinitrate, isoamyl nitrite, isosorbide dinitrate, isosorbide mononitrate, s-nitrosoglutathione, s-nitroso-n-acetylcysteine, s-nitroso-N-acetylpenicillamine, diethylamine NONOate and spermine NONOate. According to a preferred application, sodium nitroprusside (SNP) is used as an inducer.

[0053] In a preferred application of the invention, in process step of (iv) at least a carbon source selected from a group comprising sorbitol, mannitol, trehalose and glycerol is used. In addition to carbon sources such as sorbitol, mannitol and trehalose not natural AOX promoter repressors, glycerol can also be used as carbon source since SNP induction eliminates repressing property of glycerol on promoter.

[0054] SNP used in the most preferred application of the invention is used not only plant but also used in human studies as exogenous NO donor. NO donor (SNP) applied as exogenous is evidenced to reduce salt stress, heavy metal toxicity, chilling stress and herbicide damage in plants by SNP. In literature it is reported that in human exogenously-applied NO donor (SNP) is used as a drug in treatment of coronary heart disease (as vasodilator), regulation of basophil and mast cell activation, sperm motility, and synthesis of type I collagen and heat shock protein. These results all show that NO and NO donor-SNP do not have toxic effect on humans, on contrary, they can be used as drug.

[0055] In the current invention NO donors, preferably SNP increases recombinant protein expression surprisingly when used as inducer on AOX1 promoter.

[0056] SNP is not toxic, even small amounts thereof is functional in industrial scale, does not cause storing problem and is not flammable, and because of these properties its preferability is increased. In addition, use of SNP eliminates problem of breaking of sodium alginate beads and enables sodium alginate-immobilized P. pastoris cells to produce extracellular recombinant protein in continuous cultures.

[0057] On the other hand, with use of SNP as inducer, there is no need for any genetic intervention (regulation, mutation, over expression or knock-down etc) on promoter region and transcription factors for expression of heterologous gene. Therefore, essential contribution to reduce workload and cost is provided. The fact that SNP applied as a NO donor is cheap and performs heterologous gene induction at even very low concentrations also reveals that the invention can be applied at low cost on an industrial scale.

[0058] The most importantly, with use of SNP, much higher protein production can be achieved in comparison to methanol induction. According to heterologue gene expression analysis conducted, SNP induction showed similar results in the presence of glycerol compared to only methanol induction (no significant difference, p>0.05), in the presence of sorbitol a higher gene expression level was observed with SNP induction (FIG. 1). Therefore, with the method disclosed under the invention, workload and cost are reduced as well as recombinant protein quantity is considerably increased.

[0059] In a study conducted under scope of the invention, to produce recombinant protein, recombinant P. pastoris cells (constructed in our laboratory) carrying “prepromelittin protein” gene of Apis mellifera (honey bee) in its genome and producing extracellularly this protein were used. With the method of the invention, it was observed that extracellular protein production was achieved in groups, where SNP was used as inducer (at various concentrations of 200, 400, 600 and 800 nanomoles) for induction of promoter region and sorbitol was as carbon source.

[0060] In another study performed under scope of the invention, human PINX1 (hPINX1) protein under AOX1 promoter control by adding SNP in 800 nM concentration was produced intracellularly with P. pastoris. According to Western blot analysis result of this study, it was seen that SNP induced hPINX1 protein production.

[0061] As a result, method of the invention enables the conversion of human, plant, animal and microorganism genes to extracellular and intracellular recombinant proteins (insulin, growth factors etc.).