人LL-37与干扰素α2a密码子的优化及其在毕赤酵母中的融合表达

The Codon Optimization and Fused Expression of LL-37 and IFN-α2a in Pichia pastoris GS115

通过密码子优化,在毕赤酵母中高效表达人LL-37与IFN-α2a融合蛋白。先按P.pastoris密码子偏好性对LL-37与IFN-α2a的原始密码子进行了改造,并在二者之间加上Gly Gly Gly Gly Ser的柔性连接接头,人工合成设计的新序列,最后通过p PIC9K载体将其整合入P.pastoris GS115的基因组,构建出重组菌株GS115LI。利用遗传霉素浓度梯度筛选出两株高拷贝的GS115LI1和GS115LI2菌株。对这两株菌经发酵诱导后的发酵液进行SDS-PAGE检测、抗病毒活性检测和抗菌活性检测,证明重组株既能够成功表达出LL-37与IFN-α2a的融合蛋白,而且该融合蛋白成功保留了抗菌肽与干扰素的功能活性。诱导发酵后,融合蛋白的产量可达到819.1 mg/L,经盐析、疏水层析和离子交换层析分离,可得到纯度达97%的融合蛋白产物,回收率可达到46.2%,纯化后产品的效价可达2.6×108 IU/mg。经过密码子优化后,成功实现在毕赤酵母中高效表达出既有LL-37抗菌活性又具有干扰素α2a活性的融合蛋白。

To express the fussed protein of human LL-37 and IFN-α2a inPichia pastoriswith codon optimization. Recording to the codon preference ofP. pastoris, the codons of LL-37 and IFN-α2a were optimized. A flexible linker of GlyGlyGlyGlySer was placed between LL-37 and IFN-α2a. The newly scheming DNA sequence was synthesized chemically.P. pastoris GS115LI was constructed by integrating the new gene into GS115 with help of plasmid pPIC9K. With the scanning of geneticin concentration ladder, two strains, named as GS115LI1 and GS115LI2, were gotten with high copy number. With SDS-PAGE detection, anti-virus activity detection and antimicrobial activity detection of broth, the results proved GS115LI1 and GS115LI2 not only expressed the fused proteins, but also maintained the activities of LL-37 and IFN-α2a respectively. After fermentation and induction of recombinant, the productivity of fusion protein was 819.1 mg/L. With salting out, hydrophobic chromatography and ionic exchange chromatography, the purity of fusion protein was 97%, recovery rate was 46.2% and the potency of IFN was 2.6＆#215;108 IU/mg. After codon optimization, the fused protein of human LL-37 and IFN-α2a was expressed effectively inP. pastroriswith antimicrobial activity of LL-37 and antifungus activity of IFN-α2a.