Expression screening of bacterial libraries of recombinant alpha-1 proteinase inhibitor variants for candidates with thrombin inhibitory capacity

Comparison of mammalian and bacterial expression library screening to detect recombinant alpha-1 proteinase inhibitor variants with enhanced thrombin inhibitory capacity☆

“…We observed both thrombin binding to nontransfected HEK 293 cells (Gierczak et al, 2011) and to transfected cells displaying API T345R/M358R. This variant cannot form covalent complexes with thrombin (Bhakta et al, 2013;Gierczak et al, 2011;Hood et al, 1994) but it is a thrombin substrate (Gierczak et al, 2011), suggesting that transient binding of thrombin to this and other non-functional API derivatives takes place. The flow cytometric profile of the AR-API M358X transfected cell library overlapped with that of AR-API T345R/M358R, which was expected if most of the candidates did not express a functional thrombininhibitory serpin, but was indicative of an unfavorable signal to noise ratio.…”

“…Screening of libraries of bacterial colonies harbouring pBAD-H6-API plasmids varying at one or more API codons was performed as previously described (Bhakta et al, 2013). Briefly, single bacterial colonies or pools of ten colonies were used to inoculate archive plates or liquid cultures; the latter were lysed by sonication and tested by incubation with thrombin immobilized onto the wells of microtiter plates.…”

“…Briefly, single bacterial colonies or pools of ten colonies were used to inoculate archive plates or liquid cultures; the latter were lysed by sonication and tested by incubation with thrombin immobilized onto the wells of microtiter plates. Bound recombinant API variants competent for thrombin binding were subsequently detected by anti-API IgG binding and quantified by optical density following color generation by horseradish-peroxidase coupled sheep anti-human API IgG (Bhakta et al, 2013). In some experiments the time of incubation of the lysates with bound thrombin, previously set at 60 min, was varied.…”

“…Fig. 1A shows the results of screening sixteen pools of ten bacterial colonies each harbouring pBAD-API M358X plasmids using a thrombin capture assay (TCA) previously employed to screen an API P12 variable library (i.e., pBAD-API A347X) for thrombin-inhibiting variants (Bhakta et al, 2013). Six of sixteen pools exhibited optical density values indicative of thrombin-complexing activity at levels exceeding the background by at least 3-fold.…”

“…This finding suggested a strategy for selection of novel API variants by cell sorting of transfected cells acquiring the ability to bind thrombin (Gierczak et al, 2011). We also described a thrombin capture assay and its use to select functional serpin variants from pools of candidates expressed in libraries expressed in Escherichia coli (Bhakta et al, 2013). Our objectives in the current study were: (1) to adapt the mammalian cell expression of tethered API M358R for library screening;…”