Expression of active human C1 inhibitor serpin domain in Escherichia coli.
Human C1 inhibitor is a highly glycosylated serine protease inhibitor of the serpin family. The protein contains two disulfide bonds. In this study, an N-terminally truncated form of recombinant C1 inhibitor was overexpressed in Escherichia coli strains BL21(DE3) and AD494(DE3), the latter enabling the formation of disulfide bonds within the cytoplasm. With both strains, a major fraction of the recombinant protein produced appeared to be insoluble. However, the soluble fraction of lysates from strain AD494(DE3) inhibited the C1s target protease in functional assays. Recombinant C1 inhibitor produced in this strain also displayed the ability to complex with C1s in vitro. In contrast, lysates from strain BL21(DE3) displayed no C1 inhibitor activity. These data support the notion that glycosylation is not important, whereas disulfide bond formation appears to be essential for the production of an active recombinant C1 inhibitor. Thus, bacterial strains that permit the formation of disulfide bonds may represent a reliable system for the production of recombinant C1 inhibitor. However, a major obstacle to large-scale production will be to produce the protein in a soluble form. Attempts to increase the yield of soluble protein by coexpression of the GroEL/ES chaperonins resulted in an increase in solubility.
Lamark
T
T
Institute of Pharmacy, University of Tromsø, Tromsø, 9037, Norway. trondl@farmasi.uit.no
Ingebrigtsen
M
M
Bjørnstad
C
C
Melkko
T
T
Mollnes
T E
TE
Nielsen
E W
EW
D000595:Amino Acid Sequence; D001483:Base Sequence; D018835:Chaperonin 10 / Q000096:biosynthesis / Q000235:genetics; D018834:Chaperonin 60 / Q000096:biosynthesis / Q000235:genetics; D003174:Complement C1 Inactivator Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003171:Complement Pathway, Classical / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000138:chemical synthesis / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D015843:Serpins / Q000096:biosynthesis / Q000235:genetics; D012995:Solubility; D013696:Temperature
D018835:Chaperonin 10; D018834:Chaperonin 60; D003174:Complement C1 Inactivator Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015843:Serpins; D007544:Isopropyl Thiogalactoside