烟草CSN4基因的原核表达及纯化

以野生型烟草(Nicotiana tabacum)为试验材料,采用RT-PCR克隆获得CSN复合体亚基CSN4c DNA序列,基因登录号为KC866360。序列分析表明,CSN4基因ORF长为1194 bp,编码398个氨基酸,蛋白分子质量为43.78 k Da。将CSN4构建至原核表达载体,获得重组子pET28a-CSN4/BL21(DE3),通过Ni-IDA亲和层析纯化目的蛋白,SDS-PAGE和Western Blotting检测,结果显示:0.25 mM IPTG,15℃诱导16 h获得浓度为0.476 mg/m L、纯度高达95%的CSN蛋白,为后续CSN4基因功能研究奠定了基础。

The Prokaryotic Expression and Purification of Tobacco CSN4 Gene

In this study,the COP9 signal complex subunit CSN4 cDNA sequence was cloned by RT-PCR amplification from the wild type Nicotiana tabacum,accession number KC866360 in GenBank database.Sequence analysis showed that the length of open reading frame of N.tabacum CSN4 gene was 1194bp,encoding 398 amino acids,and the molecular weight of the protein is 43.78kDa.The recombinant pET28aCSN4/BL21 (DE3) had been obtained by cloning CSN4 gene into the prokaryotic expression vector pET28a.The target protein was purified by Ni-IDA affinity chromatography,and the results of SDS-PAGE and Western Blotting indicated that the recombinant strain was induced with 0.25mM IPTG at 15℃ for 16h,the target protein concentration was 0.476mg/mL and its purity was up to 95％,which was laid the foundation for further study protein-function.