Effect Of Polyamino Acids Fusion On ManA Expression And Salt Tolerance Of Pichia Pastoris

Pichia pastoris expression system is a kind of eukaryotic expression system which has been developed rapidly and widely used in recent years.In order to further improve the expression of exogenous proteins in Pichia pastoris and explore the feasibility of Pichia pastoris application in high-salt environment,the following work was carried out in this study:(1)In order to further improve the expression of Man A in Pichia,this research in Man A the C-terminal fusion of several different polymamino acid,and then respectively in Pichia expression.Flask fermentation results show that the fusion of the number of amino acids and the kinds of amino acids are the expression of secretion of Man A enzyme activity have different degree of influence.Among them,with the increase of the number of Arginines in the fusion polyamino acids,the activity of the enzyme secreted by Man A showed a trend of increasing first and then decreasing.When the C-terminal of Man A was fused with 6 Arginines,the superenzyme activity of the recombinant X33/Man A-R6 reached a maximum value of 493U/m L,which was 33% higher than that of the control strain X33/Man A(371U/m L).Seven different polyamino acids were fused at the C-terminal of Man A,compared with the control bacteria,in addition to the ecombinant X33/Man A-E6 enzyme activity(348 U/m L)declined slightly,the fusion of other 6 kinds of polyamino acid more recombinants on enzyme activity to some extent,the recombinant X33/Man A-R6 supernatant enzyme activity of the highest(493U/m L),Secondly,the recombinant X33/Man A-K6 reached the value of 431U/m L and the X33/Man A-D6 reached the value of 435 U/m L.The intracellular enzyme activity of recombinants was also analyzed when it was fermented to 120 h,and it was found that compared with the control strain,the intracellular enzyme activity of recombinants decreased in varying degrees.The above results showed that when the C-terminal of Man A was fused with a proper number of polyarginine,polylysine and polyaspartic acid,the retention of Man A in the cell could be reduced,and the enzyme activity of secretion and expression of Man A could be significantly increased.(2)In order to improve the feasibility of Pichia pastoris application in high salt environment,this study uses yeast surface display technology,using Pichia pastoris cell wall protein Gcw51 p as anchor protein,showing and expressing poly amino acids containing 1-5 Arginine on Pichia pastoris cellsurface respectively.The growth of each recombinants was monitored by shaking flask culture in the medium containing 5%,10% and 15% Na Cl respectively.The results show that,the recombinant X33/Gcw51-R2 showed the best growth in the three culture medium containing different concentrations of Na Cl.After fermentation to 120 h,in the medium containing5%,10% and 15% Na Cl,the concentration of X33/ Gcw51-R2 was 1.2 times,1.5 times and 1.2 times higher than that of the control strain X33/Gcw51.The above results indicated that the expression of polyamino acids containing 2Arginines on the cell surface could improve the salt tolerance of Pichia pastoris.