摘要
采用电转化法将已构建好的表达载体pPIC9K-GT转化到毕赤酵母(Pichina pastoris)GS115中。通过对电转化条件进行优化,达到了理想的转化效率,得到了大量转化子,并通过表型筛选及PCR鉴定,筛选到阳性转化子。对重组的P.pastoris GS115-GT进行了诱导表达,用SDS-PAGE法检测到目的蛋白质条带,证明β-1,4-半乳糖基转移酶基因(GT)在P.pastoris GS115中能够表达;用苯酚红法测定了粗酶液的活性,其比酶活为16.40 U/ml。对工程菌的发酵条件进行了初步优化,在最佳工艺下的比酶活达到30.909 U/ml,比优化前提高了88.47%。
Recombinant plasmid pPIC9K-GT was transformed into P.pastoris GS115 by electroporation.In order to achieve a high transformation efficiency,the electroporation conditions were optimized.The positive transformant was obtained after phenotype screening and PCR identification.Inducible expression of the recombinant P.pastoris GS115-GT was conducted.Target protein detected by SDS-PAGE verified β-1,4-galactosyltransferase gene was expressed solubly in P.pastoris GS115.The specific activity of β-1,4-galactosyltransferase after optimization of fermentation conditions for genetically engineered microorganim was 30.909 U/ml increased by 88.47% compared to that before optimization,which was only 16.40 U/ml.
出处
《江苏农业学报》
CSCD
北大核心
2012年第6期1367-1372,共6页
Jiangsu Journal of Agricultural Sciences
基金
国家科技部973计划(2010CB735705)
上海市科委课题(09DZ2251400)
关键词
Β-1
4-半乳糖基转移酶
电转化
诱导表达
发酵条件
优化
β-1
4-galactosyltransferase
electroporation transformation
inducible expression
fermentation condition
optimization
