The Expresstion In Vitro And Analysis Of Sfi1 Funtional Peptide In Saccharomyces Cerevisiae

Sfil protein is closely related to the cell cycle of Saccharomyces cerevisiae,and its main function is to maintain spindle assembled correctly in cell division process.It has an important role in cell division process and cell shape maintenance,but so far there are little literature to study systematacially that the Sfil protein structure and function at the molecular level.Sfilp has 21 internal repeats of LLX3F/LX2W mode, and 20 interval sequences,which they are located in the central region of the protein. The main function of the central region is to combine with yeast centrosome protein Cdc31p through the internal repeat,by which to act on the replication process of SPB.Meanwhile,the N-terminal and C-terminal regions of Sfilp do not contain the internal repeats,but bioinformatics analysis revealed that there are several possible conserved phosphoric sites of cyclin dependent kinase Cdc28.Among the sites C-terminal region has seven and N-terminal region has two.It also found that the site deletion would lead to the cell divided abnormally at the level of yeast cells.In all these ways,it plays a key role in the regulation of SPB replication function.So the project emphasized on the functional peptides to study Sfilp deeply,including conservative peptides and phosphorylation peptides.This paper discussed the functional peptides of Sfilp in the Saccharomyces cerevisiae mainly from two aspects.One side is to analyse the 21 reapeats at the central region of Sfilp,and found the 20th conservative peptide is typical to research.Then we focus on this peptide to build peptide library which consists of the wild type,the site-directed mutate,and the random mutate.We study the influence to interact with Cdc31p,and discuss the biological effect which the mutate brings to.On the other side we express the phosphorylation peptides in vitro which peptides are in the N-terminal and C-terminal of Sfilp,and it lays a foundation for the phosphorylation research.We compared the three-dimensional conformations of Sfilp-763 amino acids random mutation by the computer homology modeling,and found that the end of the L763H conformation is slightly different that the a-helix is more stretch.We cloned the wild sequence and site-directed mutational sequence,respectively using the conventional PCR and fusion PCR technology.Then We expressed the Sfi 1 peptide in the yeast cell utilizing the yeast phage display technology,and found that the best expresstion condition is temperature 20℃,IPTG concentration 0.02g/ml,induction time 24h.By the SDS-PAGE analysis,we found that Sfilp-L763H did not affect the interaction with Cdc31p.We also found that Sfilp-L763H can arrest the cell cycle in G2/M phase by the flow cytometry detecting the DNA content.We also used error-prone PCR technology to clone the coding gene bank of Sfil peptide,then cloned to pYD1 to obtain the expression vector library,finally transformed to the yeast cell to induce the peptide expresstion to obtain Sfil peptide library. We detected the effect of the interaction to Cdc31p by SDS-PAGE。In addition,We cloned the SFI1-N terminal and C-terminal sequences by the conventional PCR technology, and builded to the protein expression vector pET.Then we expressed the peptides of Sfilp-N terminal and C-terminal using the inducer IPTG in the E. coli cells of BL21.Sfilp N-terminal peptide inducing expression condition is that the induction temperature is 20℃, IPTG final concentration is 1.0mmol/L,and the timeis 4h;the Sfi 1 p-C-terminal peptide inducing expression condition is that the induction temperature is 30℃,IPTG final concentration is 1.0mmol/L,and the induction time is 8h.Sfilp N-terminal peptide and C-terminal peptide expressed in vitro successfully,and it lays the foundation for studing the Sfilp-N and C-terminal peptide phosphorylation mechanism.