Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation
Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation
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DOI：
10.1111/j.1365-2672.2005.02572.x
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发表时间：
2005-01-01
影响因子：
4
通讯作者：
Sarvas, M
中科院分区：
文献类型：
--
作者：
Vitikainen, M;Hyyryläinen, HL;Sarvas, M
目的：探讨通过工程细胞因子影响胞外蛋白质折叠和degradation.Methods和结果：影响胞外相蛋白质分泌的瓶颈组分进行基因工程改造，并通过Western印迹和酶促试验研究了它们对11种工业上感兴趣的异源蛋白质分泌的影响。PrsA脂蛋白的过度产生增强了嗜热脂肪芽孢杆菌的α-淀粉酶（4倍）和肺炎链球菌溶血素（1.5倍）的分泌。由于缺乏D-丙氨酸取代的阴离子细胞壁聚合物而增加细胞壁的净负电荷增强肺炎球菌溶血素c的分泌。1.5-fold.降低HtrA型质量控制蛋白酶的水平会对生长产生有害影响，并且不会增强分泌。百日咳毒素亚单位S1是HtrA型蛋白酶的底物，其分泌依赖于这些蛋白酶。结论：外源蛋白的分泌可以通过蛋白依赖性的方式通过参与分泌后期阶段的工程组分来增强。该研究揭示了调节分泌的易位后阶段作为提高异源蛋白产量的手段的可能性和局限性。
Aims: To explore the potential to enhance secretion of heterologous proteins in Bacillus subtilis by engineering cell factors affecting extracytoplasmic protein folding and degradation.Methods and Results: Bottleneck components affecting the extracytoplasmic phase of protein secretion were genetically engineered and their effects on the secretion of 11 industrially interesting heterologous proteins were studied by Western blotting and enzymatic assays. Overproduction of PrsA lipoprotein enhanced the secretion of alpha-amylase of Bacillus stearothermophilus (fourfold) and pneumolysin (1.5-fold). Increasing the net negative charge of the cell wall because of lack of the D-alanine substitution of anionic cell wall polymers enhanced the secretion of pneumolysin c. 1.5-fold. Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion. Pertussis toxin subunit, S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases.Conclusions: Secretion of heterologous proteins can be enhanced by engineering components involved in late stages of secretion in a protein-dependent manner.Significance and Impact of the Study: The study revealed both possibilities and limitations of modulating the post-translocational phase of secretion as a means to improve the yield of heterologous proteins.