Improvement Of Secretory Expression Of Human Serum Albumin Fusion Protein In Pichia Pastoris

Due to the potential of producing soluble, correctly folded protein with high yield, theyeast Pichia pastoris is currently one of the most widely used hosts for the expression ofheterologous proteins, especially for those recombinant human proteins. However, limitationsof expression efficiency and proteolytic degradation are often reported for many differentheterologous proteins. Secretory expression of a series of peptides genetically fused to humanserum albumin (HSA) in P. pastoris was successfully achieved in previous work of mylaboratory. The secretion levels of different HSA fusion proteins in P. pastoris were muchlower compared with HSA when using the same strain and expression vector under theoptimal cultivation condition. In addition, different HSA fusion proteins usually underwentproteolytic degradation in different degree during secretory expression. To circumvent thesedefects and improve the secretory expression of HSA fusion proteins in P. pastoris, weinvestigated the possible effects of genetic engineering the Pichia expression system of HSAfusion proteins by following three aspects:(1) To avoid the possible negative effect of methanol on the expressed HSA fusion protein,a constitutive expression vector for secretory expression of rhIL-2-HSA fusion protein in P.pastoris was constructed. The coding gene was placed in frame with the Saccharomycescerevisiae ?-factor secretion signal sequence under control of the GAP promoter. Theresulting recombinant plasmid pGAPz?A-rhIL-2-HSA was integrated into the genome of theP. pastoris GS115by electroporation. The effect of different carbon sources on secretoryexpression of rhIL-2-HSA fusion protein was evaluated in shaking flask cultures. We foundthat recombinant P. pastoris grew well and rhIL-2-HSA fusion protein was the mostefficiently secreted into the medium when using glucose as carbon source. Fed-batchfermentation strategy using glucose as carbon source for constitutive expression ofrhIL-2-HSA fusion protein was investigated in5-L bioreactor. The expression level ofrhIL-2-HSA could reach about250mg·L-1after60h fermentation. The rhIL-2-HSA fusionprotein produced by this constitutive expression system was purified and exhibited a specificbioactivity of1.040×106IU·mg-1in vitro, which is similar to the rhIL-2-HSA produced byAOX1derived expression system.(2) Several steps in the secretory pathway, such as folding within the ER and vesicletrafficking are suggested to be bottlenecks during recombinant protein secretion in yeast.Three ER resident proteins including immunoglobulin binding protein(BiP), protein disulfideisomerase (PDI) and ER oxidoreductin (Ero1), and two putative Sec1/Munc18(SM) proteins(Sec1and Sly1) involved in membrane fusion during vesicle trafficking were cloned from P.pastoris and selected as potential secretion helper factors to investigate the effects of theiroverexpression on secretory expression of HSA fusion protein rhIL-2-HSA. Constitutiveoverexpression of the five selected secretion factors did not have obvious negative effect oncell growth of the rhIL-2-HSA secreting strain. Among the five secretion helper factors,individually co-overexpression of the Ero1, PDI, BiP, Sec1, and Sly1improved the secretionlevel of rhIL-2-HSA by about120%,130%,90%,150%, and90%comparing to the controlstrain. Western blot analysis of the intracellular level of IL-2-HSA protein showed that thecontent of IL-2-HSA protein in the cytosolic protein fraction and membrane-associated protein fraction in each of the five co-overexpression strains were also increased comparing tothat in the control strain. qPCR analysis also showed that the transcription levels of someother secretion helper factor genes were increased when one secretion helper factor wasoverexpressed.(3) To alleviate the degradation of HSA fusion protein during secretory expression in P.pastoris, the five Yapsin protease genes assigned as YPS1, YPS2, YPS3, YPS7and MKC7in P.pastoris were disrupted individually or simultaneously. We then evaluated the expression of aHSA fusion protein (HSA-ADAM15) in various P. pastoris protease disruption strains (yps1?,yps2?, yps3?, yps7? and mkc7?). Unfortunately, the degradation of this HSA fusion protein(HSA-ADAM15) during secretory expression was not alleviated in thesesingle-protease-deficient strains. We further constructed several multiple-protease-deficientstrains and used them as hosts for expression of HSA-ADAM15fusion protein in shake-flaskcultivation. SDS-PAGE analysis of the HSA fusion protein (HSA-ADAM15) expressed bythese multiple-protease-deficient strains showed that double disruptants (yps1?yps2? andyps1?yps3?) did not reduce the amount of degradation bands d1(~66kDa) and d2(~45kDa)comparing to the parental strain GS115. The amount of intact HSA-ADAM15in GBD4(yps1?yps2?yps3?) was about133%of that in strain GS115, and the amount of degradationbands d1and d2were reduced by about50%and52%, respectively, comparing to that instrain GS115. These results showed that the multiple disruptants of Yapsin protease genes arepotential industrially valuable hosts for secretory expression of HSA-ADAM15fusion protein.In addition, among these putative GPI-linked aspartyl proteases, we unexpectly found thatdisruption of PpYPS7gene conferred the yps7? mutant cell increased resistance to cell wallperturbing reagents Congo red (CR), Calcofluor white (CW) and SDS. Quantitative analysisof cell wall components showed lower content of chitin and increased amounts of?-1,3-glucan. Further staining the cell with Calcofluor white demonstrated that disruption ofPpYPS7gene caused a reduction of the chitin content in lateral cell wall. Consistently, TEMshowed that the inner layer of mutant cell wall, mainly composed of chitin and ?-1,3-glucan,was much thicker than that in parental strain GS115. Additionally, yps7? mutant alsoexhibited increased resistance to KCl and NaCl compared with parental strain GS115. Thiscould be due to the dramatically elevated intracellular glycerol level in yps7? mutant. Theseresults suggested that PpYPS7's function is involved in cell wall integrity and response toosmotic stress.