【作者】 史明；

【导师】 李钰；

【作者基本信息】 哈尔滨工业大学 ， 生化与分子生物学， 2007， 硕士

【摘要】 p18基因全长3 232 bp,含有2个外显子,编码区位于第1外显子内编码522bp,GC含量高达90%。以往研究表明,p18基因与多种肿瘤的发生有关。生物信息学分析,P18蛋白分子量约18.9kD,存在4次跨膜的结构域。通过胶体金定位分析,确定P18蛋白亚细胞定位于内质网和线粒体。由于p18基因编码区高GC含量和P18蛋白的高疏水性,p18基因的可溶性表达存在着很大的困难。本研究根据生物信息学分析P18蛋白的疏水性和二级结构特点,构建了p18基因不同的截短体,尝试p18基因截短体的可溶性表达。根据外源基因与一些易表达的序列共翻译能大幅提高表达水平,将目的基因构建到谷胱甘肽转硫酶(GST)融合表达的载体中形成重组体,在大肠杆菌和酵母中摸索最佳表达条件,最终表达出携带目的蛋白的GST融合蛋白。进一步利用谷胱甘肽-琼脂糖亲和层析对目的蛋白进行纯化。实验结果表明,转入重组体pGEX-6P-1-p18-280、pGEX-6P-1-p18-397、pGEX-6P-1-p18-433的大肠杆菌Rosetta(DE3)菌株在37℃、1mM IPTG浓度下能够大量表达,但经可溶性分析得知,这些表达的蛋白绝大部分为包涵体。优化诱导条件后,转入上述重组体的大肠杆菌Rosetta(DE3)菌株在16℃、0.1mM IPTG浓度下获得了可溶性表达。转入重组体pYES2-GST-p18的酵母INVSc1在30℃、2%半乳糖诱导72h条件下,经纯化也得到了可溶性的GST-P18融合蛋白,但其表达量远没有在大肠杆菌中表达量高。综合分析上述结果可以得知,在大肠杆菌中低温诱导GST融合蛋白是获得重组蛋白可溶性表达的有效方法,但表达量受目的基因的长度、GC含量和二级结构等因素影响。而重组体在酿酒酵母中进行诱导表达虽然可获得较长片段的基因表达,但其表达量过低。为满足进一步实验的需求,仍需不断优化外源基因在酵酒酵母中的表达条件。更多还原

【Abstract】 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.更多还原