Chow Dominic C, Dreher Matthew R, Trabbic-Carlson Kimberly, Chilkoti Ashutosh
Department of Biomedical Engineering, Duke University, Box 90281, Durham, North Carolina 27708, USA.
Biotechnol Prog. 2006 May-Jun;22(3):638-46. doi: 10.1021/bp0503742.
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 a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides.
弹性蛋白样多肽（ELPs）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相比，这些氨基酸在ELPs中的比例异常高，我们推测这些氨基酸的细胞内池可能会被选择性耗尽，并在表达过程中限制蛋白质产量。在本研究中，我们通过监测表达带有绿色荧光蛋白（GFP）标签的ELP的大肠杆菌的细胞生长和蛋白质表达动力学，研究了培养条件和单个培养基成分如何影响蛋白质产量。通过确定超表达方案产生高融合蛋白产量的潜在原理，我们通过添加甘油和某些氨基酸（如脯氨酸和丙氨酸）进一步提高了蛋白质产量，发现氨基酸浓度和所用基础培养基的类型对这种有益效果有强烈影响。令人惊讶的是，除了ELPs中丰富的氨基酸外，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能提高蛋白质产量。与常用的Luria-Bertani培养基相比，在摇瓶培养中使用改良培养基和优化培养条件，蛋白质产量提高了36倍，达到了1.6 g/L的显著水平，这也使融合蛋白的成本降低了8倍。据我们所知，这是从摇瓶培养的大肠杆菌中纯化的ELP融合蛋白的最高产量。本研究还提出了一种提高其他ELP融合蛋白和重复多肽产量的有用策略。
