Coexpression Of Polyproteins Mediated By 3C Fusion Protein

As so far, a myriad of studies where single gene has been successfully introduced into or manipulated in yeast, animal and plant cells or whole organisms have been reported. However, for the reason of the limited effects of single genes in changing some valuable metabolism or interconnected pathways and treating the complex important characters, co-expression of multiple genes has been increasingly placed more emphasis by many researchers. Several conventional techniques, termed ’stacking’ or ’pyramiding’, are involved to allow transgenes to be combined in a single individual. However, these strategies suffer from several critical problems which limit their utilization.Self-processing polyproteins using protease from virus is to link proteins in tandem by recognizing sequence of protease in a single open reading frame giving a single polyproteins, which will be regulated by one promoter and take advantage in transgenic technology and process. After translation, polyproteins are processed into constituent polypeptides. NIa protease from tobacco etch virus （TEV） is one of the proteases involved in virus cleavage system. Because of its specificity, 3C protease can also be used to process target proteins in vivo. Self-processing polyproteins using protease is a form of post-translational modification, which means the chloroplast transit peptide produced by the cleavage is valid to sort and target while the signal peptide in secretary pathway produced by the cleavage can not be recognized by the SRP and would be futile.The 55 amino acid sequence of N-terminal region ofβNAC of yeast and the 118 amino acid sequence of N-terminal region of TF of E. coli contains the NAC signature binding domain respectively. In this study, in order to co-express polyproteins in Pichia pastoris and target the GFP protein produced by the self-processing polyproteins using protease to different subcellular location, we synthesized the DNA fragment of NH and TH motif using gene splicing by overlap extension, and fused them to 3C protease, we also designed 9 kinds of single-promoter-driven constructs, in which GFP was supposed to be targeted to cytoplasm, ER or extracellular compartment. We linearized the plasmid of these constructs by Sac I and transformed them into Pichia pastoris by electroporation. Selected by G418 geneticin, we acquired multiple copies insertion transformants. Then some of them were chosen randomly to be cultivated in BMGY until the value of OD₆₀₀ of yeast was 4-6, and the compounds were collected and expressed in BMMY inducing by methanol for 6 hours. At last, the expression of polyproteins and the form of GFP was detected by western blot using the anti-His and anti-GFP as the first antibody.The results indicated that, among the constructs intent of targeting GFP to the cytoplasm, the ratio between GFP and intact polyproteins was significantly higher in pN3CG and pT3CG than in p3CG, which revealed that 3C could be targeted to the nascent polypeptide exit tunnel of ribosome by the motif of NH and TH, and the recognition sequence of protease could be cleaved by 3C before it folded to a higher structure, which led to a more effective cleavage. It also revealed that the TF from E.coli and the NAC from Pichia pastoris may employ a conserved ribosome binding domain to bind to ribosome.Among the constructs intent of targeting GFP to the extracellular compartment, we acquired the some naive GFP without signal peptide in the constructs of p3CAG. The fact that the signal sequenceαF, produced by the post-translational cleavage, could still be recognized and targeted testified that SRP-independent pathway in directing and translcoation to ER existed in Pichia pastoris. In the constructs of pT3CAG and pN3CAG, the signal sequence produced by co-translational cleavage could emerge in the nascent polypeptide exit tunnel as the N-terminal region of polypeptide and be recognized by the signal recognition particle （SRP）. However, their translocation results were similar to the p3CAG’s, which implied that the pathway specificity in choosing SRP dependent or SRP independent is conferred by the signal sequences.Among the constructs intent of targeting GFP to the ER, KAR-HDEL was a well characterized substrate, by which there were two targeting pathways to the ER operating side-by-side in yeast, SRP-dependent co-translational translocation route and SRP-independent post-translational translocation route. The fact that some GFP in the form of KAR-GFP is acquired in p3CKGH by post-translational cleavage suggested that they were targeted to the ER by SRP-independent post-translational translocation route, while in the cotranslational cleavage of pN3CKGH and pN3CKGH two forms of GFP, namely, KAR-GFP and GFP were acquired, which indicated that the two different pathways to the ER might account for the disparity in excising the signal sequence KAR by the protease in ER.In this thesis, the possible significance of co-expression of polyproteins in identifying whether the signal sequences were SRP-indepentdent in directing and translcoation to ER was described. In addition, the strategy and mechanics of binding to the ribosome of some molecular chaperons and the measures and potential function of ribosome modification was discussed.