Abstract：
The secretion efficiency of a heterologous protein in E. coli is mainly dictated by the N-terminal signal peptide fused to the desired protein. In this study, we aimed to select and introduce mutations into the - 1, - 2 and - 3 positions of the gIII signal peptide (originated from filamentous phage fd Gene III) fused to the N-terminus of the human growth hormone (hGH), and study its effect on the secretion efficiency of the recombinant hGH into the periplasmic space of E. coli Top10. Bioinformatics software such as SignalP-5.0 and PrediSi were employed to predict the effects of the mutations on the secretion efficiency of the recombinant hGH. Site-directed mutagenesis was applied to introduce the desired mutations into the C-terminus of the gIII signal peptide. The periplasmic expression and the secretion efficiency of the recombinant hGH using the native and mutant gIII signal peptides were compared in E. coli Top10 under the control of araBAD promoter. Our results from bioinformatics analysis indicated that the mutant gIII signal peptide was more potent than the native one for secretion of the recombinant hGH in E. coli. While our experimental results revealed that the mutation had no effect on hGH secretion. This result points to the importance of experimental validation of bioinformatics predictions.
摘要：
大肠杆菌中异源蛋白的分泌效率主要由与所需蛋白融合的N-末端信号肽决定。在这项研究中,我们旨在选择并将突变引入到gIII信号肽（源自丝状噬菌体fd基因III）的-1，-2和-3位置，与人生长激素（hGH）的N端融合，并研究其对重组hGH向大肠杆菌Top10周质间隙分泌效率的影响。使用生物信息学软件如SignalP-5.0和PrediSi来预测突变对重组hGH分泌效率的影响。应用定点诱变以将所需突变引入gIII信号肽的C端。在araBAD启动子控制下,在大肠杆菌ToplO中比较使用天然和突变gIII信号肽的重组hGH的周质表达和分泌效率。我们的生物信息学分析结果表明，突变体gIII信号肽比天然肽更有效地在大肠杆菌中分泌重组hGH。虽然我们的实验结果表明该突变对hGH分泌没有影响。该结果指出了生物信息学预测的实验验证的重要性。
