Protein Expression and Purification 22, 349–358 (2001) doi:10.1006/prep.2001.1445, available online at http://www.idealibrary.com on Expression of Active Human C1 Inhibitor Serpin Domain in Escherichia coli Trond Lamark,* ,1 Monica Ingebrigtsen,* Camilla Bjørnstad,* Tarja Melkko,† Tom E. Mollnes,* , ‡ and Erik W. Nielsen* , § *Institute of Pharmacy, University of Tromsø, 9037 Tromsø, Norway; †Department of Clinical Chemistry, University of Oulu, Oulu, Finland; and ‡Department of Immunology and Transfusion Medicine and §Department of Anesthesiology, Nordland Central Hospital, Bodø, Norway Received February 6, 2001, and in revised form March 13, 2001; published online June 14, 2001 serine proteases C1s and C1r from the classical path- Human C1 inhibitor is a highly glycosylated serine way—and probably MASP-1 and MASP-2 in the lectin protease inhibitor of the serpin family. The protein con- pathway—of complement. This mainly hinders the acti- tains two disulfide bonds. In this study, an N-terminally vation of C4 and C2. C1-Inh also is a major inhibitor truncated form of recombinant C1 inhibitor was over- of kallikrein and of activated factor XII of the contact expressed in Escherichia coli strains BL21(DE3) and system, thereby limiting production of the vasoactive AD494(DE3), the latter enabling the formation of disul- peptide bradykinin. fide bonds within the cytoplasm. With both strains, a C1-Inh is a member of the serine protease inhibitor major fraction of the recombinant protein produced (serpin) family (2), which also comprises important se- appeared to be insoluble. However, the soluble fraction rum protease inhibitors such as prothrombin, 2-mac- of lysates from strain AD494(DE3) inhibited the C1s roglobulin, 2-antiplasmin, 1-antitrypsin, PAI-1, and target protease in functional assays. Recombinant C1 PAI-2. In a reaction between a protease and a serpin inhibitor produced in this strain also displayed the family inhibitor, the protease binds to the inhibitor and ability to complex with C1s in vitro. In contrast, lysates cleaves it at a specific recognition site within the so- from strain BL21(DE3) displayed no C1 inhibitor activ- called reactive center loop. This initiates a conforma- ity. These data support the notion that glycosylation tional change resulting in the deformation of the prote- is not important, whereas disulfide bond formation ase so that it becomes “trapped” to the serpin in a cova- appears to be essential for the production of an active lent and enzymatically inactive complex (3). recombinant C1 inhibitor. Thus, bacterial strains that C1-Inh is a highly glycosylated protein with a total permit the formation of disulfide bonds may represent weight of 104,000 Da (2). The protein backbone of 478 a reliable system for the production of recombinant C1 inhibitor. However, a major obstacle to large-scale amino acids constitutes half of this mass. A partial production will be to produce the protein in a soluble removal of sialic acid or galactose groups did not impair form. Attempts to increase the yield of soluble protein the functional activity of C1-Inh in vitro (4) and the by coexpression of the GroEL/ES chaperonins resulted biological role of the carbohydrate groups is not known. in an increase in solubility.  2001 Academic Press Most of the carbohydrate groups are located on a 97- amino-acid-long N-terminal domain that is unique for C1-Inh. This domain is apparently not needed for com- plex formation, since N-terminally truncated C1-Inh C1 inhibitor (C1-Inh) is an important inhibitor of (residues 98–478) produced in COS-1 cells retained its several plasma cascade systems (1 and references biological activity (5). The rest of the protein consists therein). It is the only known inhibitor of the activated of the serpin domain, which is characteristic for all serpins. In C1-Inh, the serpin domain contains two di- 1 To whom correspondence should be addressed at the Institute sulfide bonds, which were recently shown to be required of Pharmacy, University of Tromsø, 9037 Tromsø, Norway. Fax: 47 77646151. E-mail: trondl@farmasi.uit.no. for the protein to be stabilized in the metastable state 1046-5928/01 $35.00 349 Copyright  2001 by Academic Press All rights of reproduction in any form reserved.