Yield, solubility and conformational quality of soluble proteins are not simultaneously favored in recombinant Escherichia coli.
Many enzymes or fluorescent proteins produced in Escherichia coli are enzymatically active or fluorescent respectively when deposited as inclusion bodies. The occurrence of insoluble but functional protein species with native-like secondary structure indicates that solubility and conformational quality of recombinant proteins are not coincident parameters, and suggests that both properties can be engineered independently. We have here proven this principle by producing elevated yields of a highly fluorescent but insoluble green fluorescent protein (GFP) in a DnaK- background, and further enhancing its solubility through adjusting the growth temperature and GFP gene expression rate. The success of such a two-step approach confirms the independent control of solubility and conformational quality, advocates for new routes towards high quality protein production and intriguingly, proves that high protein yields dramatically compromise the conformational quality of soluble versions.
Martínez-Alonso
Mónica
M
Department of Genetics and Microbiology, Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain.
García-Fruitós
Elena
E
Villaverde
Antonio
A
D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013696:Temperature
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C063842:dnaK protein, E coli