Production and secretion of human interleukin 6 into the periplasm of Escherichia coli: efficient processing of N-terminal variants of hIL6 by the E. coli signal peptidase.
We have developed a system for expressing human interleukin 6 into the periplasmic space of Escherichia coli. The method is based on the expression of the hIL6 gene under the control of the regulatory signals of plasmid pINIII-OMPA3, i.e., lpp-lac promoter and the E. coli OMPA ribosome binding site and leader sequence. Since microheterogeneity is known to occur in the amino end of the cytokine, we tested different 'natural' versions of the protein, and we found that the secretion process was only efficient when the N-terminal amino acid was not proline. In flask experiments this procedure yields about 8-10 mg of biologically active hIL6 per liter.
Barthelemy
I
I
Biotechnology Research Center, Antibióticos Farma, S.A. Erbamont Group, Madrid, Spain.
González de Buitrago
G
G
Carreiro
C
C
Roncal
F
F
Pérez-Aranda
A
A
Márquez
G
G
Barbero
J L
JL
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D014644:Genetic Variation; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012270:Ribosomes / Q000378:metabolism; D012697:Serine Endopeptidases
D015850:Interleukin-6; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C008705:octamethyl pyrophosphoramide; D004247:DNA; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase