Ultra-high expression of a thermally responsive recombinant fusion protein in E. coli.
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.
Chow
Dominic C
DC
Department of Biomedical Engineering, Duke University, Box 90281, Durham, North Carolina 27708, USA.
Dreher
Matthew R
MR
Trabbic-Carlson
Kimberly
K
Chilkoti
Ashutosh
A
D000596:Amino Acids / Q000494:pharmacology; D002244:Carbon / Q000494:pharmacology; D002452:Cell Count; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004549:Elastin / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007700:Kinetics; D009584:Nitrogen / Q000494:pharmacology; D009842:Oligopeptides / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D000596:Amino Acids; D017077:Culture Media, Conditioned; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002244:Carbon; D004549:Elastin; D009584:Nitrogen