Bae Jung-Hoon, Sung Bong Hyun, Seo Jeong-Woo, Kim Chul Ho, Sohn Jung-Hoon
Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Industrial Microbiology and Bioprocess Research Center, Korean Research Institute of Bioscience and Biotechnology, Jeongeup, 56212, Republic of Korea.
Appl Microbiol Biotechnol. 2016 Dec;100(24):10453-10461. doi: 10.1007/s00253-016-7722-2. Epub 2016 Jul 13.
Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.
与融合伙伴一起表达蛋白质可提高产量并简化纯化过程。我们开发了一种新型融合伙伴，以改善在酵母中原本分泌不佳的异源蛋白质的分泌。酿酒酵母的VOA1（YGR106C）基因编码液泡ATP酶的一个亚基。我们发现，C末端截短的Voa1p会大量分泌到培养基中，即使与很少分泌的异源蛋白质如人白细胞介素-2（hIL-2）融合时也是如此。对C末端截短的Voa1p进行缺失定位，确定了一个由28个氨基酸组成的亲水性肽（HL肽），它负责增强靶蛋白的分泌。添加了一个纯化标签和一个蛋白酶切割位点，以便将HL肽用作多功能融合伙伴。通过表达和纯化各种异源蛋白质来测试该系统的实用性。在许多情况下，与该肽融合的靶蛋白产量显著增加，融合蛋白可通过亲和色谱直接纯化。通过体外处理去除融合伙伴，通过将样品重新应用于亲和色谱来纯化完整的蛋白质。
