The Regulation Of Pichia Pastoris Surface Display Based On Flocculation Functional Domain Of Flo1

Pichia pastoris express system as a eukaryotic expression system has developed rapidlyin recent years. P. pastoris express system has many advantages such as high expression ofheterologous protein, low cost of cultivation, so it has been widely applied. Flo1displaysystem from Saccharomyces cerevisiae is an effective yeast display system, it has C and Nterminal two display mode. FFD is the N terminal flocculation domain of Flo1, using the FFDdisplay foreign proteins can avoid the interference of enzyme with active center in C terminal,so it is meaningful to display enzyme with active center in C terminal used FFD. This paper ismainly to study the factors which influence FFD expression of heterologous.(1) Construct the evaluation system of FFD surface displayed systermIn this study, FFD was used to display Enhance Green Fluorescent Protein (EGFP) andCandida antarctica lipase B (CALB). An inducible promoter AOX1was used to constructrecombinant strains GS115/pKFS-EGFP, GS115/pKFS-EGFP-CALB andGS115/pKFS-CALB-EGFP. By inducing the expression of heterologous protein, fluorescenceintensity, protein concentration and enzyme activity were detected, protein concentration inthe supernatant and the fluorescence intensity of EGFP were correlated. The formula offluorescence intensity and protein concentration was used to calculate the amount of proteindisplayed by fluorescence intensity. The calculated GS115/pKFS-EGFP showed the fusionprotein was up to9×104protein per cell, while GS115/pKFS-EGFP-CALB andGS115/pKFS-CALB-EGFP showed the fusion protein for up to2×104protein per cell.(2) The study of different concentrations of mannose influence the display ability of FFDIn order to express heterologous protein without methanol, constitutive promoter GAPwas used to construct recombine strain GS115/pPICZA-GAP-FS-EGFP. TheGS115/pPICZA-GAP-FS-EGFP was cultivate in different mannose concentration (2%,3%,4%,5%,6%) YPM medium (YPD medium as control) and detected the EGFP fluorescenceintensity. The increase of mannose concentration of the cultivate medium will improve theexpression of heterologous proteins displayed by FFD display system. The highest displayfluorescence intensity was1.71×107RFU/mL when mannose concentration is6%, thefluorescence intensity decrease when concentration of mannose decrease, the minimum fluorescence intensity is1.71×107RFU/mL of2%mannose. The appropriate amount ofmannose in medium will improve display effect.(3) The study of different pH of cultivate medium influence the display ability of FFDThe recombine strain GS115/pPICZA-GAP-FS-EGFP was cultivated and detect thefluorescence intensity of EGFP. Research of pH show that within the pH5.0, pH6.0, pH7.0and pH8.0pH, the optimum in pH for FFD display system was pH5.0, the fluorescenceintensity of EGFP GS115/pPICZA-GAP-FS-EGFP shows up to5.5×106RFU/mL, thedisplay fluorescence intensity of pH5.0is about1.125,1.29,1.5times of pH6.0, pH7.0, pH8.0.The recombinant strains GS115/pKFS-EGFP, GS115/pKFS-EGFP-CALB andGS115/pKFS-CALB-EGFP constructed before cultivated in different pH (pH5.0, pH6.0,pH7.0) and the detection of EGFP fluorescence intensity and CALB enzyme activity. Found aconsistent trend with recombinant GS115/pPICZA-GAP-FS-EGFP results obtained before, inpH5.0FFD display system has the highest expression of heterologous protein effect.(4) The study of different deletion units of FS repeat unit A influence the display abilityof FFD display systermThis research studied the deletion of different repeat units of FS repeat unit A influencethe display ability of FFD. Using nonspecific overlap extension PCR construct fourrecombine strains GS115/pKFS-EGFP(ΔA6-13), GS115/pKFS-EGFP(A5-13),GS115/pKFS-EGFP(A4-13) and GS115/pKFS-EGFP(A3-13) which deleted89,10,11repeatunits of repeat unit A. By detecting the fluorescence intensity of EGFP, we found that with theincrease of deleted units of repeat unit A, the number of EGFP display by FFD will reduce.Positive control GS115/pKFS-EGFP has the highest fluorescence intensity of1.98×108RFU/g, the fluorescence intensity of recombinant strains which deleted8,9,10and11repeatunits of repeat unit A were1.26×107RFU/g,1.13×107RFU/g,0.98×107RFU/g and0.86×107RFU/g. As the number of units deleted from repeat unit A increased, the display ability ofFFD system will decrease. The study also found that as the deleted units increase theflocculation of the recombine strain will increase.