Expression and export in Escherichia coli of fusion proteins containing carboxy-terminally located honeybee prepromelittin.
The aim of this work was to express a eukaryotic pre-protein in Escherichia coli so that it could be obtained intact, without cleavage, by bacterial leader peptidase. To this end, cDNA coding for honeybee prepromelittin was ligated to the 3' end of genes coding for truncated forms of either Protein A or beta-galactosidase (beta-Gal) under the control of inducible promoters, with an oligonucleotide coding for the Factor Xa cleavage site at the junction between the two proteins. The Protein A fusion was expressed in good yield, and about 80% of it formed inclusion bodies. The prepromelittin section of the Protein A fusion caused some export of the intact fusion protein to the growth medium. The prepromelittin beta-Gal fusion was expressed in low yield and became associated with the E. coli cytoplasmic membrane. Its expression was toxic to E. coli. Thus, the synthesis of a full-length eukaryotic pre-protein in E. coli is best achieved when the fusion protein forms inclusion bodies.
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Department of Surgery, St. George's Hospital Medical School, London, UK.
Liu
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Austen
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B
D000818:Animals; D001516:Bees; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008555:Melitten / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D008555:Melitten; C016694:prepromelittin; D001616:beta-Galactosidase