Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

Abstract

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2–P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352–356 (P7–P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7–P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of “designer serpins” with novel reactivity and/or specificity.

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

“…This strategy preserved 16 of 20 possible amino acids as possibilities between P7-P2 in our "BNN" API M358R library, at the cost of not sampling Phe, Tyr, Trp, or Cys residues at these positions. The variant with the strongest signal in the thrombin capture assay that was found on screening of this library, TLSATP, inhibited thrombin 2.9-fold more rapidly than API M358R, surpassing P7-P3 AAFVS or DITMA as the most rapid API variant obtained from expression library screening efforts to date (Scott et al, 2014). TLSAT fit the "P7-Not Aromatic/P6-Hydrophobic/P5-T or S/P4-Hydrophobic/P3-Not Aromatic" loose consensus previously defined in our P7-P3 screening efforts (Scott et al, 2014).…”

“…The variant with the strongest signal in the thrombin capture assay that was found on screening of this library, TLSATP, inhibited thrombin 2.9-fold more rapidly than API M358R, surpassing P7-P3 AAFVS or DITMA as the most rapid API variant obtained from expression library screening efforts to date (Scott et al, 2014). TLSAT fit the "P7-Not Aromatic/P6-Hydrophobic/P5-T or S/P4-Hydrophobic/P3-Not Aromatic" loose consensus previously defined in our P7-P3 screening efforts (Scott et al, 2014). Like DITMA or AAFVS, the TSLATP improvement in the rate of thrombin inhibition did not come at the cost of an increased reaction stoichiometry.…”

“…5. Lysates containing API M358R exhibited a gradual increase in thrombin-bound API reactivity over 60 min, while two API M358R variants with additional mutations previously shown to enhance their rate of thrombin inhibition by 2- (Scott et al, 2014) to 19-fold (Sutherland et al, 2006) exhibited more rapid rates of thrombin binding that plateaued over time, and API T345R/M358R exhibited minimal, background levels of binding (Fig. 5).…”

“…Although we did not observe enrichment for any specific P1 variant, including M358R, in the cell-sorting screen, we cannot exclude the possibility that such enrichment might have emerged following multiple rounds of HEK 293 display-mediated screening. However, since we have already described a multiple round method for selection of thrombin inhibitory variants of API M358R, T7 phage display (Scott et al, 2014), we did not pursue this possibility, but instead compared bacterial expression screening of the same hypervariable P1 library.…”

“…denotes "not significant" (p > 0.05). library hypervariable at P2-P1, which yielded only Pro-Arg (Scott et al, 2014), and consideration of serpin RCL sequences (Gettins, 2002). Of the 27 human serpins known to function as protease inhibitors, 13 contain Arg at P1 and two Lys; of the serpins that inhibit thrombin, only heparin cofactor II lacks a P1 Arg (Gettins, 2002).…”