Order of fusions between bacterial and mammalian proteins can determine solubility in Escherichia coli.
We made fusions between Escherichia coli maltose-binding protein (MBP) and the mammalian aspartic proteinases pepsinogen or procathepsin D. When MBP was at the N-terminus, the fusions were soluble in E. coli. When the order was reversed, the chimeric proteins formed inclusion bodies. The data suggest that the solubility of fusion proteins is controlled by whether the protein domains emerging first from the ribosome normally fold into soluble or insoluble states. The soluble MBP-aspartic proteinase fusions were stable but proteolytically inactive. MBP-pepsinogen, however, was efficiently renatured from 8 M urea in vitro, suggesting that the E. coli cytoplasm does not support folding of the mammalian partner protein to the native state. Thus, inclusion body formation may be the consequence, rather than the cause, of non-native folding in vivo, and in E. coli soluble proteins may fold into states different from those reached in vitro.
Sachdev
D
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Research Service, Audie L. Murphy Memorial Veterans' Administration Medical Center, San Antonio, Texas, USA.
Chirgwin
J M
JM
D018528:ATP-Binding Cassette Transporters; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D002479:Inclusion Bodies; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010435:Pepsinogens / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D004792:Enzyme Precursors; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010435:Pepsinogens; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D016282:Aspartic Acid Endopeptidases; C058345:procathepsin D; D002402:Cathepsin D