Design of a solubilization pathway for recombinant polypeptides in vivo through processing of a bi-protein with a viral protease.
An artificial maturation pathway for increasing the solubility in vivo of recombinant proteins overproduced in Escherichia coli is reported, which is based on the proteolytic processing of viral polyproteins. The gene product of interest is expressed as a fusion to a heterologous moiety (i.e. the maltose binding protein, MalE) in order to increase the overall solubility of the hybrid. The hinge region between the two fusion partners contains a cleavage site for the NIa protein, a very specific protease from the plum pox potyvirus, as well as an affinity tag. After production, the soluble hybrid is cleaved in vivo by the protease, that is encoded by a plasmid harboured by a specialized E.coli host. The released protein remains soluble and can be purified from cell extracts by means of an affinity tag (a poly-His group) that becomes present after the cleavage. The solubilization and purification of XylR, a xylene-responsive transcriptional factor of Pseudomonas, with this method are reported.
Pérez-Martin
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J
Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid, Spain.
Cases
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de Lorenzo
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D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011549:Pseudomonas / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics*; D012995:Solubility; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C074322:XylR protein, Pseudomonas; C461599:maltose transport system, E coli; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses