Secretion of heterologous gene products to the culture medium of Escherichia coli.
Different constructs containing fragments of the Staphylococcal protein A gene have been introduced in Escherichia coli and the effect on expression and translocation of the various heterologous gene products have been studied. By reversing the orientation of the different protein A gene constructions in the plasmid vector, a dramatic 20-fold difference in expression was obtained, accompanied with secretion of the gene product to the culture medium. Similar results were obtained by "heat-shock" treatment of the E.coli host cells. These results suggest the presence in the protein A gene of a stress induced promoter, functional in E.coli. The system was used to efficiently secrete a fusion protein consisting of a protein A fragment and human insulin-like growth factor I (IGF-I) to the culture medium of E.coli HB101. The fusion protein was purified from the culture medium by IgG affinity chromatography in a one-step procedure giving more than 95% yield.
D003470:Culture Media; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D010957:Plasmids; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000302:isolation & purification
D003470:Culture Media; D013205:Staphylococcal Protein A; D004262:DNA Restriction Enzymes