Ultra-high expression of a thermally responsive recombinant fusion protein in E-coli
Ultra-high expression of a thermally responsive recombinant fusion protein in E-coli
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DOI：
10.1021/bp0503742
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发表时间：
2006-06-01
影响因子：
2.9
通讯作者：
Chilkoti, Ashutosh
中科院分区：
文献类型：
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作者：
Chow, Dominic C.;Dreher, Matthew R.;Chilkoti, Ashutosh
弹性蛋白样多肽（ELP）是基于重组肽的生物聚合物，含有富含甘氨酸、缬氨酸、脯氨酸和丙氨酸的重复序列。由于与其他细胞蛋白质相比，ELP中这些氨基酸的比例异常大，我们假设这些氨基酸的细胞内库可以被选择性耗尽并限制表达过程中的蛋白质产量。在这项研究中，我们研究了如何培养条件和个别培养基成分影响蛋白质产量通过监测细胞生长和蛋白质表达动力学的E。表达用绿色荧光蛋白（GFP）标记的ELP的大肠杆菌。通过确定由超表达方案产生的上级融合蛋白产量的基本原理，我们通过添加甘油和某些氨基酸如脯氨酸和丙氨酸进一步提高了蛋白产量，并发现氨基酸浓度和所用基础培养基的类型强烈影响这种有益效果。令人惊讶的是，除了在ELP中丰富的氨基酸之外的氨基酸，例如天冬酰胺、天冬氨酸、谷氨酰胺和谷氨酸，即使在营养丰富的培养基中也提高了蛋白质产量。与常用的Luria-Bertani培养基相比，采用改进的培养基和优化的培养条件进行摇瓶培养，蛋白产量提高了36倍，达到1.6g/L的显著水平，同时融合蛋白的成本降低了8倍。据我们所知，这是从E.大肠杆菌在摇瓶中培养。这项研究还表明
Elastin-like polypeptides (ELPs) are recombinant peptide-based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fraction of these amino acids in ELPs as compared to other cellular proteins, we hypothesized that intracellular pools of these amino acids can be selectively depleted and limit protein yields during expression. In this study, we examined how culture conditions and individual medium components affect protein yields by monitoring cell growth and protein expression kinetics of E. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining the underlying principles of superior fusion protein yields generated by the hyperexpression protocol, we further improved protein yields through the addition of glycerol and certain amino acids such as proline and alanine and found that amino acid concentrations and the type of basal medium used strongly influenced this beneficial effect. Surprisingly, amino acids other than those that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamic acid, also enhanced protein yields even in a nutrient-rich medium. Compared to commonly used Luria-Bertani medium, the protein yield was improved by 36-fold to the remarkable level of 1.6 g/L in shaker flask cultures with a modified medium and optimized culture conditions, which also led to a 8-fold reduction in the cost of the fusion protein. To our knowledge, this is the highest yield of an ELP-fusion protein purified from E. coli cultured in shaker flasks. This study also suggests