Zhu Fuxiang, Liu Zelong, Qu Huige, Xin Xiaolin, Dong Hongxin, Liu Xiangqin
Life Science College of Ludong University, Yantai 264025, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Jul;25(7):1101-6.
We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.
我们利用内含肽介导的蛋白质反式剪接研究了凝血因子VIII重链和轻链在大肠杆菌中的连接。将一个缺失B结构域的因子VIII（BDD-FVIII）基因在符合剪接所需保守残基的Ser1657之前分成重链和轻链的两半，然后分别与含有106和48个氨基酸的N端部分（称为Int-N）和C端部分（称为Int-C）的分裂型小Ssp DnaB内含肽编码序列融合。将这两个融合基因构建到原核表达载体pBV220中。通过诱导重组蛋白表达，在SDS-PAGE凝胶上显示出一条明显的蛋白带，其大小与预测的BDD-FVIII蛋白大小一致。使用因子VIII特异性抗体进行的蛋白质印迹证实，这条蛋白带是由蛋白质反式剪接产生的BDD-FVIII。这表明BDD-FVIII的重链和轻链可以通过Ssp DnaB内含肽介导的蛋白质反式剪接有效地连接。这些结果为鼓励我们正在进行的以内含肽作为手段在携带因子VIII基因的双AAV载体中克服血友病A基因治疗中单个AAV载体包装大小限制的研究提供了证据。
