The Study Of P18 Gene Expression In Escherichia Coli And Saccharomyces Cerevisiae

P18 gene is 3 232 bp in length, containing two exons, code area located in exon 1 coding 522bp, GC content as high as 90%. Past research has shown that p18 gene involves in the variety of tumor occurrence. Bioinformatics analyses that subcellular localization of P18 protein is majorly in endoplasmic reticulum and mitochondria. The molecular weight of P18 protein is about 18.9kD. There are four transmembrane domains. Because of high GC content and high hydrophobicity, soluble p18 protein is difficult to express.According to bioinformatics analysis of the P18 protein hydrophobic and secondary structural features, we construct p18 truncated gene and try to express soluble truncated P18 protein. As exogenous genes coexpress with some genes that can easily express, expression level will increase. Constructing recombinant protein by inserting the target gene into glutathione S-transferase (GST) fusion expression vector, in E. coli and yeast searching the best method to express, finally express the target protein GST fusion protein. Further, purify the target protein using affinity chromatography.Experimental results show that E.coli Rosetta(DE3), the transformed recombinant pGEX-6P-1-p18-280 or pGEX-6P-1-p18-397 or pGEX-6P-1-p18-433, cultivating at 37°C , inducing in 1mM IPTG medium can express fusion protein, but being informed by the analysis of solubility, the majority of these proteins are inclusion body. After optimization induced conditions, E.coli Rosetta (DE3), transformed the same recombinant, cultivating at 16℃, inducing in 0.1mM IPTG medium can obtain soluble expression. S.cerevisiae INVSc1, transfected recombinant pYES2-GST-p18, cultivating at 30℃, induced at 2% galactose for 72h, can obtain soluble P18-GST fusion proteins. But the fusion protein expressed in S. cerevisiae is far less than being expressed in E.coli.These results show that it is an effective means to express soluble GST fusion protein in E. coli at low temperature. However, the expression of target genes affect by the factors such as the length of GC content and the secondary structure. The recombinant could be expressed in S.cerevisiae, but the amount of fusion protein is too low. To meet the demand for further experiments, it still will be optimized expression condition in S. cerevisiae.