The Study In The Influence Of The Chaperones And The Signal Peptides On The Expression Level Of The Secretory Protein In Pichia Pastoris

Pichia pastoris is the preferential engineering bacteria which has been expandively used for its advantages of relative high-expression level , graduately perfect fermentation technology and easily manipulation of protein purification. But some serious problems, such as depression of the expression level, the activity cut down even not at all and the new protein or peptide aggregation and sedimentation, emergencying concomitant the new heterogeny protein explored in this yeast expression system, and result in interfere the generalization applying of yeast. So,it is necessary to research the bioprocessing of translation, translocation, folding and secretory and the other essentional factors of the protein expression in yeast, in order to solve these problems.As well as known, the secretory signal peptides and the chaperones play a essential role in the process of the protein synthesis and translocation. Signal peptides is a amino acid sequence which conduct the nascent peptide translocate to the signal recognition protein on the membrane of ER, then guide the protein secretory to the definite location. Therefore, signal peptides worked in all the protein translocation. Meanwhile, the chaperones are a large of accessory molecules that reside in the whole process of the protein synthesis, they take part in the translocation, folding and unfolding, disaggregation and degradation of mistake proteins ensure the normal conformation and activity of the protein products.The subject of our study are the chaperones and the signal peptides,and this study characterize in the first consider the chaperones that locate in different cell positions and also the relationship between the chaperons and the signal peptides, we expect to understand the interactions among the chaperones selves and to the signal peptides,finally we may identify the details of mechanism in which the chaperones and the signal peptides play, and then proposed the feasible program to submit e the expession level.We choose EXG1 as the report protein which is an endogenous enzyme of pichia pastoris. We study the optimize expression strategy in pichia pastoris according to the influence of the chaperones that located in cytoplasma, the membrane and ventric of the ER and the signal peptides to the effect of the EXG1 expression level in yeast GS115 and GS200. Fllowing is the plane of the experimental. First,adding the post-translation translocation signal peptides,αMF andαpre, and the co-translation translocation signal peptide, Bip, to the N terminal of the EXG1 in order to construct the plasmid of EXG1/αMF-pPIC9 , EXG1/αpre-pPIC9, EXG1/Bip-pPIC9 ,EXG1-pPIC9. As the same time, insert the chaperone genes to pBLArg-IX. After those, explore the expression level of the report protein EXG1 led with different signal peptides in pichia pastoris GS115(His-), and then coexpress the chaperones with the EXG1 adding different signal peptides in pichia pastoris GS200(His-/Arg-). Induce expression of the EXG1 in the shake flasks by 0.5% methanol, and then evaluate the influence of the chaperones and the signal peptides on the EXG1 expression level.As the results show, the first, different signals affect obviously the expression capacity of the EXG1, specially,the expression level of EXG1/Bip which has a co-translation translocation signal is 2 times to the EXG1 with self signal,but the level of EXG1/Bip is still lower than EXG1/Αmf and EXG1/αpre. The second, the chaperones that sec61α, sec61βand Ydj1, Ssa1, Hsp104 which located in the membrane of ER and the cytoplasm respectively have no affection to the different EXG1. Although, Bip,Ero1,PDI and Hac1 obviously promote different EXG1, boost the EXG1/αMF and EXG1/αpre up to 2.6-3.8 times,and the EXG1-Bip about 20-45%,and no affection on the EXG1 with self signal peptide. As show above, in yeast cell,co-translation translocation signal peptide confirmLy promote EXG1 expression, and the chaperones in ER are important factors to EXG1 expression. Meanwhile,Bip and Ero1 could elevate obviously the EXG1/αMF, EXG1/αpre and EXG1/Bip under the effect of Hac1 who is a messenger molecule. Finally we still can't certificate the chaperones preferred any signal peptide.