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首页> 外文期刊>Applied and Environmental Microbiology >Improved Secretory Production of Recombinant Proteins by Random Mutagenesis of hlyB, an Alpha-Hemolysin Transporter from Escherichia coli
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Improved Secretory Production of Recombinant Proteins by Random Mutagenesis of hlyB, an Alpha-Hemolysin Transporter from Escherichia coli

机译：通过随机诱变hlyB（一种来自大肠杆菌的α-溶血素转运蛋白）提高重组蛋白的分泌产生

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摘要

Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA218) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 × 104 E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23°C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA218 fusion protein at 23 and 30°C but unexpectedly not at 37°C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA218 was detected only in the L448F mutant (AE104F) at 23°C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA218 fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37°C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.
机译：具有α-溶血素（HlyA）C端信号序列的融合蛋白已知是由大肠杆菌中的HlyB-HlyD-TolC易位子分泌的。我们旨在通过对hlyB和hlyD进行随机诱变来建立有效的Hly分泌表达系统。枯草杆菌蛋白酶E和HlyA信号序列的融合蛋白（HlyA218）用作评估分泌效率的标记蛋白。通过筛选超过1.5×104个大肠杆菌JM109转化子（其hlyB和hlyD基因已通过易错PCR诱变），我们成功地分离出了两个枯草杆菌蛋白酶E分泌活性水平高27和15倍的突变体比野生型在23°C时要高。这些突变体在23和30°C时也表现出增加的分泌单链抗体-HlyA218融合蛋白的活性水平，但出乎意料的是在37°C时却没有，表明这种改善似乎取决于低温。发现一个突变体（AE104）在HlyB和HlyD中均具有七个点突变，并且HlyB中的L448F取代负责改善分泌活性。另一个突变体（AE129）在HlyB中进行了单个氨基酸取代（G654S）。仅在23°C的L448F突变体（AE104F）中检测到c-Myc-HlyA218的分泌，而在任何温度下在野生型中均未观察到分泌。此外，对于PTEN-HlyA218融合蛋白，AE104F的分泌活性水平比野生型在37°C时高10倍。该结果表明，AE104F的分泌活性提高并不总是取决于低温。

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