人CTLA4胞外区突变体在毕赤酵母中的高效表达

High-level expression of mutant CTLA4 extracellular domain in Pichia Pastoris

目的 根据毕赤酵母密码子偏好,设计并改造人CTLA4胞外区cDNA,构建毕赤酵母分泌型表达载体,以实现人CTLA4胞外区蛋白在毕赤酵母中的高效表达。方法利用PCR定点突变技术,将人CTLA4胞外区cDNA中的毕赤酵母低频使用密码子突变成高频使用密码子而不改变其氨基酸序列;经DNA序列测定证实后插入毕赤酵母高拷贝表达载体pPIC9K a分泌信号下游,SacⅠ线性化后电转GS115;G418梯度筛选转化菌;PCR鉴定目的基因整合;甲醇诱导表达,SDS-PAGE、Western blotting、肽质量指纹等鉴定表达蛋白。结果成功地对人CTLA4胞外区cDNA进行了定点突变,该突变体在毕赤酵母中能成功表达CTLA4胞外区蛋白。结论 为高效表达人CTLA4胞外区蛋白及其功能研究和临床应用奠定了基础。

Objective To get high-level expression of human cytotoxic T lymphocyte associated antigen-4 (CT-LA4) extracellular protein in Pichia Pastoris by designing and modification of CTLA4 extracellular domain coding sequence and construction of a secretion expression vector based on the codon usage bias of Pichia Pastoris. Methods The low frequency usage codon was changed into high frequency usage synonymous codon without changing the ami-no acid sequence by PCR-based site-directed mutagenesis. After DNA sequence analysis, the high frequency usage synonymous codon was inserted into the Pichia Pastoris expression vector pPIC9K. Following electroporation of the Sac I -linearized DNA into the Pichia Pastori strain GS115 (his-4) , His + transformants were recovered and plated in medium containing increasing concentrations of the G418. The target gene integration was identified by PCR and the expression was induced by methanol. The induced protein expression was identified by SDS-PAGE, Western blotting and peptide mass fingerprint analysis. Results The human CTLA4 extracellular domain coding sequence was successfully modified as expected. The mutant expression vector successfully expressed CTLA4 extracellular protein at high level in the fermentation supernatant. Conclusion This study provides a foundation for the studies of high-level expression of human CTLA4 extracellular protein, its functions and clinical application.