Serpin Phage Display: The Use of a T7 System to Probe Reactive Center Loop Libraries with Different Serine Proteinases

Abstract

Phage display is a protein engineering approach that involves construction of libraries of variant proteins displayed on the surface of bacteriophage as capsid fusion proteins and their screening for binding and inhibitory function through the use of bait proteins. Recently, we adapted a commercially available T7 phage display system to create phage-displayed serpin libraries hypervariable in up to five positions in their reactive center loop (RCL). The RCL is a key determinant in serpin specificity, the relationship between the structure of a given serpin and which target proteinase(s) it inhibits. In this chapter, we describe protocols to assess the feasibility of this method for different serpin/proteinase combinations and share experience with this technology gathered in the course of studying two serpins and multiple proteinases with this powerful iterative screening approach.

Stepwise Reversion of Multiply Mutated Recombinant Antitrypsin Reveals a Selective Inhibitor of Coagulation Factor XIa as Active as the M358R Variant

“…Given the more conservative nature of the I360T substitution, our previous finding that an I360T/P361Q substitution enhanced thrombin reactivity of AAT M358R (45), and the diametrically opposed decreases in thrombin reactivity of AAT-RC and AAT-RC-2 and increases in FXIa reactivity of these proteins, it is likely that I356E and P361E (P3') are primarily responsible for the desirable properties of AAT-RC-2 as a selective FXIa inhibitor. While there is some precedent for FXIa favoring Glu at P3, in that FXIa cleaves the bond following EPR in an amyloid beta protein precursor (46), in engineered activatable hirudin-based thrombin inhibitors (47,48), and in a chloromethyl ketone chromogenic substrate with specificity for FXIa (49), no precedent exists for a favorable interaction with Glu at P3'.…”

Therapeutic SERPINs: Improving on Nature

“…This probably relates to the non-linear structure of the RCL loop. To overcome this obstacle, others have performed high throughput SERPIN screening studies with the T7 phage display system (75,76). While this method allowed to find thrombin inhibitors that are twice more potent that a1AT-Pittsburgh, their specificity toward other proteases remains to be investigated.…”

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

“…78 This system can be exploited to vary different portions of the RCL and/or to probe with different proteinases via positive or negative selection. 127…”

“…Our group also used a T7 phage display system to fully randomize the P7‐P3 residues of α 1 ‐AT M358R, and to select two novel variants that enhanced the rate of thrombin inhibition by twofold via directed evolution: DITMA and AAFVS (compared to wild type FLEAI residues) . This system can be exploited to vary different portions of the RCL and/or to probe with different proteinases via positive or negative selection …”