Ultra-High Expression of a Thermally Responsive Recombinant Fusion Protein in E. coli
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- 作者：Dominic C. Chow ; Matthew R. Dreher ; Kimberly Trabbic-Carlson ; and Ashutosh Chilkoti
- 刊名：Biotechnology Progress
- 出版年：2006
- 出版时间：June 2006
- 年：2006
- 卷：22
- 期：3
- 页码：638 - 646
- 全文大小：462K
- 年卷期：v.22,no.3(June 2006)
- ISSN：1520-6033
文摘
Elastin-like polypeptides (ELPs) are recombinant peptide-based biopolymers that contain repetitivesequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fractionof these amino acids in ELPs as compared to other cellular proteins, we hypothesized thatintracellular pools of these amino acids can be selectively depleted and limit protein yieldsduring expression. In this study, we examined how culture conditions and individual mediumcomponents affect protein yields by monitoring cell growth and protein expression kinetics ofE. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining theunderlying principles of superior fusion protein yields generated by the hyperexpression protocol,we further improved protein yields through the addition of glycerol and certain amino acidssuch as proline and alanine and found that amino acid concentrations and the type of basalmedium used strongly influenced this beneficial effect. Surprisingly, amino acids other thanthose that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamicacid, also enhanced protein yields even in a nutrient-rich medium. Compared to commonly usedLuria-Bertani medium, the protein yield was improved by 36-fold to the remarkable level of 1.6g/L in shaker flask cultures with a modified medium and optimized culture conditions, whichalso led to a 8-fold reduction in the cost of the fusion protein. To our knowledge, this is thehighest yield of an ELP-fusion protein purified from E. coli cultured in shaker flasks. Thisstudy also suggests a useful strategy to improve the yields of other ELP fusion proteins andrepetitive polypeptides.