蜡样芽胞杆菌M22 Mn-SOD基因的分子改造及在毕赤酵母中的表达  

Optimization of Mn-containing Superoxide Dismutase (Mn-SOD) Gene from Bacillus cereus M22 and Its Expression in Pichia pastoris

作　　者：徐艳[1] 王琦[1] 梅汝鸿[1] 

机构地区：[1]中国农业大学植物病理学系,北京100193

出　　处：《农业生物技术学报》2008年第5期881-885,共5页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2006AA10A211)资助

摘　　要：根据毕赤酵母(Pichia pastoris)密码子的偏好性,以氨基酸序列不变为原则,对源于蜡样芽胞杆菌(Bacillus cerues)M22的Mn-SOD基因进行分子改造,设计、合成了新的基因序列Mn-SOD-2。构建酵母表达载体pPICZαA/Mn-SOD-2,并整合至毕赤酵母GS115染色体。结果表明,所构建的重组体经0.5%甲醇诱导表达后,Native-PAGE检测证实有清晰单一活性条带;SDS-PAGE检测证实重组蛋白的分子量24kD。酶活分析表明,外源蛋白的活性较改造前增加了2.2倍,且表达稳定性良好。According to the biased codons usage ofPichia pastoris, the gene of Mn-SOD from Bacillus cerues M22 was optimized and synthesized on the premise of no modification of the amino acid sequence. A recombinant plasmid pPICZo：A/Mn-SOD-2 was constructed and integrated into the genome of Pichia pastoris GS 115 by electroporation after linearization. The positive transformant was filtrated by zeocin resistance and PCR with specific primers. Analysises of Native-PAGE and SDS-PAGE showed that SOD had obvious activity and a relative molecular weight was about 24 kD as expected, indicating that the recombinant Mn-SOD-2 protein was successfully expressed in P. pastoris. The maximum activity of the recombinant with codons optimized reached 228 U/mL after inducing, which was nearly 3.2 times of the original strain, and showed the upstanding expression stability.

关 键 词：锰超氧化物歧化酶(Mn-SOD) 毕赤酵母 基因改造 表达 

分 类 号：S188[农业科学—农业基础科学]