摘要
目的在毕赤酵母中表达Thermomonospora fusca纤维素酶Cel6A。方法提取Thermomonospora fusca基因组DNA作模板,PCR扩增纤维素酶Cel6A基因,克隆到毕赤酵母表达载体pGAPZαA上,用电击法将线性Cel6A-pGAPZαA核酸片段转化毕赤酵母X-33株,克隆PCR鉴定法筛选阳性转化株,培养传代。选择稳定株,培养3d后取培养液上清,用His-BindQuick Cartridge300提取表达的重组Thermomonospora fusca纤维素酶Cel6A。结果一步His-Bind Quick Cartridge300层析可提取电泳纯的重组Thermomonospora fusca纤维素酶Cel6A,SDS-PAGE分析显示相对分子质量约为60000,收率为200mg/L,用羧甲基纤维素法测活性为500U/mg,用滤纸法测活性为1U/mg。结论已在毕赤酵母中成功表达了Thermomonospora fusca纤维素酶Cel6A。
Objective To express the cellulase Ce16A of Themomonospora fusca in Pichia pastoris. Methods Amplify cellulase Ce16A gene by PCR using the extracted genomic DNA of Themomonosporafusca as a template. Clone the amplified gene into vector pGAPZαA and transform to Pichia pastoris X-33 strain by electroporation. ,Screen the positive transformants by colony PCR method for subculture. Select stable cell strains and culture for 3 d. Collect the culture supernatant and purify the expressed recombinant cellulase Ce16A of Themomonosporafusca by His-Bind Quick Cartridge 300 chromatography. Results Electrophoretically pure recombinant cellulase Ce16A of Themomonosporafusca was obtained by one-step His-Bind Quick Cartridge 300 chromatography. SDS-PAGE showed that the relative molecular weight of expressed product was 60 000. The recovery rate of expressed protein was 200 mg/L. The activity of expressed protein was 500 U/mg by earboxymethyl cellulose method and 1 U/mg by filter paper method. Conclusion Recombinant cellulase Ce16A of Thernornonosporafusca was successfully expressed in Pichia pastoris.
出处
《中国生物制品学杂志》
CAS
CSCD
2006年第6期578-579,582,共3页
Chinese Journal of Biologicals
