Expression of Active Human C1 Inhibitor Serpin Domain in Escherichia coli

Lamark, Trond; Ingebrigtsen, Monica; Bjørnstad, Camilla; Melkko, Tarja; Mollnes, Tom Eirik

doi:10.1006/prep.2001.1445

Cited by 32 publications

(16 citation statements)

References 23 publications

(3 reference statements)

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“…In contrast, our finding that C1inhD97 forms a transient complex with the inactive C1s Ser617Ala mutant does not imply that the reaction does not proceed to a covalent complex when active C1s is the target. In keeping with previous functional investigations (11,18), the experiments carried out in the current study using C1s and C1r-LP as target proteases provide further evidence that the serpin domain of C1 inhibitor contains the structural determinants required for mediating protease inhibitory activity and that the N-terminal extension of C1 inhibitor is not implicated in this function.…”

Section: Discussionsupporting

confidence: 89%

“…This strategy reveals that, although mutation of the former two residues has little effect on protein production, removal of the oligosaccharide linked to Asn 330 results in a dramatic decrease in the expression yield, suggesting that this particular carbohydrate may play a role in the folding and/or the stability of the serpin domain. Previous attempts to prevent overall glycosylation of rC1 inhibitor or its serpin domain were made using expression in a bacterial system (18) or in the presence of tunicamycin (11,15). Although these preparations were found to retain protease inhibitory activity, inhibition of glycosylation was reported to significantly decrease the expression yield (15) or to yield a major fraction of the recombinant material in insoluble form (18).…”

Section: Discussionmentioning

confidence: 99%

“…Previous attempts to prevent overall glycosylation of rC1 inhibitor or its serpin domain were made using expression in a bacterial system (18) or in the presence of tunicamycin (11,15). Although these preparations were found to retain protease inhibitory activity, inhibition of glycosylation was reported to significantly decrease the expression yield (15) or to yield a major fraction of the recombinant material in insoluble form (18). In contrast, it is noteworthy that a latent form of the C1 inhibitor serpin domain was crystallized after enzymatic removal of N-linked carbohydrates by Endoglycosidase H (19).…”

Section: Discussionmentioning

confidence: 99%

“…Active C1 inhibitor was also expressed at high yield in Pichia pastoris and purified (17). Various constructs corresponding to the serpin domain of C1 inhibitor were also produced in COS-1 cells (10), Escherichia coli (18), and recently in P. pastoris (19). Using the latter recombinant protein, the crystal structure of the C1 inhibitor serpin domain has been solved, revealing a latent conformation with the uncleaved reactive center loop inserted into a seven-stranded antiparallel b-sheet (19).…”

mentioning

confidence: 99%

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Functional Characterization of the Recombinant Human C1 Inhibitor Serpin Domain: Insights into Heparin Binding

Rossi

Bally

Ancelet

et al. 2010

The Journal of Immunology

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Variants of the human C1 inhibitor serpin domain containing three N-linked carbohydrates at positions 216, 231, and 330 (C1inhΔ97), a single carbohydrate at position 330 (C1inhΔ97DM), or no carbohydrate were produced in a baculovirus/insect cells system. An N-terminally His-tagged C1inhΔ97 variant was also produced. Removal of the oligosaccharide at position 330 dramatically decreased expression, precluding further analysis. All other variants were characterized chemically and shown to inhibit C1s activity and C1 activation in the same way as native C1 inhibitor. Likewise, they formed covalent complexes with C1s as shown by SDS-PAGE analysis. C1 inhibitor and its variants inhibited the ability of C1r-like protease to activate C1s, but did not form covalent complexes with this protease. The interaction of C1 inhibitor and its variants with heparin was investigated by surface plasmon resonance, yielding KD values of 16.7 × 10−8 M (C1 inhibitor), 2.3 × 10−8 M (C1inhΔ97), and 3.6 × 10−8 M (C1inhΔ97DM). C1s also bound to heparin, with lower affinity (KD = 108 × 10−8 M). Using the same technique, 50% inhibition of the binding of C1 inhibitor and C1s to heparin was achieved using heparin oligomers containing eight and six saccharide units, respectively. These values roughly correlate with the size of 10 saccharide units yielding half-maximal potentiation of the inhibition of C1s activity by C1 inhibitor, consistent with a “sandwich” mechanism. Using a thermal shift assay, heparin was shown to interact with the C1s serine protease domain and the C1 inhibitor serpin domain, increasing and decreasing their thermal stability, respectively.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Functional Characterization of the Recombinant Human C1 Inhibitor Serpin Domain: Insights into Heparin Binding

Rossi

Bally

Ancelet

et al. 2010

The Journal of Immunology

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“…Subsequently, rhC1-INH was produced in higher quantities by transfected Escherichia coli strains, but only a small percentage was found to be functionally active [40].…”

Section: Recombinant Human C1-inhmentioning

confidence: 99%

Therapeutic Approaches in Hereditary Angioedema

Antoniu

2011

Clinic Rev Allerg Immunol

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Hereditary angioedema (HAE) is characterized by acute attacks of edema with multiple localizations, the laryngeal angioedema being the most potentially lethal. In HAE, C1-INH impairments cause episodic increase in kallikrein activity leading to attacks of angioedema. Several therapies have recently become available to treat or to prevent HAE attacks, and others are under evaluation for this indication. Plasma-derived C1-INH, bradykinin receptor antagonists (icatibant), kallikrein inhibitors (ecallantide), or recombinant C1-INH is authorized on the market for HAE attack therapy or prophylaxis. Some of these compounds can be used exclusively to treat HAE attacks, whereas others can also be used as prophylactic therapies. Such therapies, although not available worldwide, can improve disease outcome due to their different mechanisms of action.

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