Secretion of active truncated CD4 into Escherichia coli periplasm.
A truncated molecule containing the first 183 amino acid residues of the HIV-1 receptor, CD4, was made by periplasmic secretion in Escherichia coli. The signal sequence from the E. coli proteins OmpA, PhoA, or OmpF was fused to the truncated CD4, under the control of either the trp or the lac promoter. The processed material secreted into the periplasm reacted with monoclonal antibodies and exhibited binding activity to the HIV-1 envelope protein gp120. Not all of the processed product was recovered in the periplasm by osmotic shock, suggesting that either the material aggregated in the periplasm or, during secretion, the molecule assumed some transient conformation that interfered with its translocation across the inner membrane. A mutation in prlA (a gene involved in secretion) increased the level of processing, suggesting that secretion of a heterologous protein in E. coli can be optimized by manipulating the host secretion apparatus.
Molecular Biology, Upjohn Company Kalamazoo, Michigan 49007.
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Rockenbach
M J
MJ
Dupuis
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TW
Pitts
C K
CK
Marschke
C S
CS
Tomich
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D007785:Lactose / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014158:Transcription, Genetic; D014364:Tryptophan / Q000235:genetics
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D001425:Bacterial Outer Membrane Proteins; D015699:HIV Envelope Protein gp120; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014364:Tryptophan; D007785:Lactose