Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli.
Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis alpha-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250-700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins.
Qian
Zhi-Gang
ZG
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering, BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, KAIST, Daejeon, Republic of Korea.
Xia
Xiao-Xia
XX
Choi
Jong Hyun
JH
Lee
Sang Yup
SY
D000469:Alkaline Phosphatase / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D003470:Culture Media / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D020738:Leptin / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D021382:Protein Sorting Signals; D020543:Proteome / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D029968:Escherichia coli Proteins; D020738:Leptin; D021382:Protein Sorting Signals; D020543:Proteome; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D000516:alpha-Amylases; D004765:Enteropeptidase