<p>α<sub>1</sub>-proteinase inhibitor (α<sub>1</sub>-PI) is the most abundant serine protease inhibitor (serpin) in plasma. The α<sub>1</sub>-PI M358R mutant exhibits greatly increased rates of thrombin inhibition compared to wild type α<sub>1</sub>-PI, which predominantly inhibits neutrophil elastase. M358R (P1) lies at the reactive centre (P1-P1’) bond of the reactive centre loop (RCL) of α<sub>1</sub>-PI, cleaved by cognate proteases as they become trapped in the serpin-type inhibitory complex. The relationship between RCL structure and serpin inhibitor function is incompletely understood and has not been subjected to saturation mutagenesis. α<sub>1</sub>-PI M358R is a less potent inhibitor of thrombin than natural thrombin-inhibitory serpins, suggesting room for engineered improvement into an antithrombotic protein drug.</p> <p>Phage display is a powerful tool for screening mutant protein libraries, but only one serpin (PAI-1) has previously been mutated and expressed in this manner. In this study the T7Select10-3b (Novagen) phage display system was used to express α<sub>1</sub>-PI variants and PAI-1, fused to the first 348 residues of the T7 10B coat protein. Following confirmation that α<sub>1</sub>-PI M358R retained inhibitory activity when fused to T7Select10-3b phage, this system was used to express a library of α<sub>1</sub>-PI mutant proteins with all possible codon combinations at positions P2 (P357) and P1 (M358) (441 mutants). The library was biopanned using a novel technique in order to amplify only the α<sub>1</sub>-PI P2P1 mutants capable of forming stable complexes with thrombin. The P357/M358R mutant was the only P2P1 mutant enriched, indicating that the α<sub>1</sub>-PI M358R protein has the optimal P2P1 sequence for thrombin inhibition.</p> <p>A second T7Select10-3b library of α<sub>1</sub>-PI mutant proteins was generated to identify the optimal sequence at positions P7 through to P3 (amino acids 352-356) for thrombin inhibition. The P2 and P1 positions were maintained at P357/M358R, while all possible codon combinations at positions P7 through to P3 were represented (>4.08 million mutants). The library was biopanned using the protocol developed for the P2P1 library, before sequences were inserted into an <em>E. coli</em> expression vector and α<sub>1</sub>-PI M358R P7-P3 mutants were screened for thrombin inhibitory activity. 80 individual colonies were screened, yielding 22 unique P7-P3 mutants with thrombin inhibitory activity greater than the M358R RCL sequence. The consensus observed in sequences with improved activity matched thrombin’s known substrate specificity and also general RCL trends: P7-Not Aromatic/P6-Hydrophobic/P5-T or S/P4-Hydrophobic/P3-Not Aromatic.</p> <p>Kinetic characterization of selected mutants with improved thrombin inhibitory activity yielded two mutants, P7-P3 sequence DITMA and AAFVS, with a second order rate constant of 1.0 x 10<sup>6</sup> M<sup>-1</sup>s<sup>-1</sup>. This represents a >2-fold increase in the rate of thrombin inhibition versus α<sub>1</sub>-PI M358R. Both the DITMA and AAFVS mutants were found to have a lower stoichiometry of inhibition compared to α<sub>1</sub>-PI M358R, indicating that an improved thrombin inhibitory mechanism was also enriched during biopanning.</p> <p>These findings suggest that based on the scaffold of the α<sub>1</sub>-PI protein, improved thrombin inhibitory activity can be engineered and selected via phage display. Additionally, this work represents a proof-of-principle for the application of this system to screen libraries of up to 10 million mutants in order to better engineer serpins towards a desired activity.</p> / Master of Health Sciences (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/13367 |
| Date | 10 1900 |
| Creators | Scott, Benjamin M. |
| Contributors | Sheffield, William P., Junop, Murray, Bramson, Jonathan, Medical Sciences (Thrombosis & Haemostasis & Atherosclerosis) |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
Page generated in 0.0023 seconds