大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接

Separate expression of ABCA1 and ligation mediated by Ssp DnaE intein in Escherichia coli BL21(DE3

摘要:

摘 要：【目的】利用Ssp DnaE intein的蛋白质反式剪接技术研究在大肠杆菌中对ABCA1基因表达产物的连接作用。【方法】将ABCA1的cDNA于满足剪接所需的保守性氨基酸Cys978密码子前断裂为N端和C端两部分，分别与天然存在的反式作用Ssp DnaE intein的123个氨基酸的N端和36个氨基酸的C端编码序列融合，构建到原核表达载体pET-28a(+)。转化感受态大肠杆菌BL21(DE3)细胞，诱导表达后观察重组蛋白的表达和ABCA1的连接。【结果】转化菌经IPTG诱导表达，SDS-PA

Abstract:

Abstract: [Objective] By exploring Ssp DnaE intein-catalyzed protein trans-splicing we aimed to investigate the ligation of expression product of ATP-binding cassette transporter A1(ABCA1) gene in E. coli. [Methods] The ABCA1 cDNA was broken into two halves of N-part and C-part before Cys978 codon which meets the splicing required conserved residue, and then fused to 123 and 36 amino acid-containing N terminal and C terminal coding sequences of Ssp DnaE intein naturally occurring trans-splicing intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pET-28a(+). After transformation into E coli BL21(DE3) cells followed by induction the expression of recombinant proteins and the ligation of ABCA1 were observed. [Results] Through IPTG induction for expression of recombinant protein it displayed an obvious protein band as predicted size of ABCA1 on SDS-PAGE gel. Western blotting using His-Tag specific antibody confirmed that this protein band is trans-spliced ABCA1. [Conclusion] The data demonstrated that Ssp DnaE intein can efficiently catalyze the ligation of ABCA1 providing an evidence for our ongoing study on ABCA1 gene transfer by a dual AAV vector system to circumvent AAV volume limitation in gene therapy of Tangier disease which resulted from ABCA1 gene mutations.