Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space.
Two enzymes, the secreted Staphylococcus aureus nuclease A and the Klenow fragment of the cytoplasmic Escherichia coli DNA polymerase I, were fused, at the genetic level, to MalE, the periplasmic maltose-binding protein of E. coli, or to a signal-sequence mutant. The hybrid proteins were synthesized in large amounts by E. coli under control of promoter malEp. The synthesis was repressed with glucose and could be totally switched off in a malT mutant strain. The hybrid between MalE and the nuclease was exported into the periplasmic space. Several criteria demonstrated that a fraction of the hybrid chains with the Klenow polymerase was exported to the periplasm in a signal-sequence-specific manner and ruled out the possibility of a membrane leakage. The hybrid with the Klenow polymerase was not exported and remained in the cytoplasm when carrying a tight signal-sequence mutation in its MalE portion. The hybrid proteins were purified in one step by affinity chromatography on cross-linked amylose. Most of the hybrid chains in the periplasm but only a fraction of those in the other cell compartments had their MalE portion correctly folded. The nuclease and the Klenow polymerase had their full specific activities in the purified hybrids. The potential of MalE as a vector for the production, export and purification of desirable proteins in E. coli is discussed.
Bedouelle
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H
Unité de Biochimie des Régulations Cellulaires (Centre National de la Recherche Scientifique Unité Associée 1129), Institut Pasteur, Paris, France.
Duplay
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P
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D004256:DNA Polymerase I / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals; C461599:maltose transport system, E coli; D004256:DNA Polymerase I; D003851:Deoxyribonucleases