Expression and Characterization of Protein Chimera with Embedded Activated Protein C Generation

Increasing demand of medical implants has lead researchers to develop biomaterial surfaces that offer improved performance and lower thrombogenecity. There have been attempts to model biomaterial surfaces after the native endothelium as it represents an optimal non-thrombotic surface. Typically, when biomaterials are contacted with blood, they often invoke the activation of clotting cascades. Thus, to overcome surface mediated thrombotic events, proteins and molecules with anticoagulant attributes of the endothelium have been immobilized onto biomaterial surfaces. Protein C (PC) plays a main role in blood coagulation and acts as an anticoagulant when it is converted into activated protein C (APC). Activation of PC can be initiated only by thrombin (TR)-thrombomodulin (TM) complex with a relatively slower reaction, and is enhanced 20-fold when EPCR, the co-factor to PC, is also present. The objective of this thesis is to generate a chimeric protein that includes the functional domains of both EPCR and TM in an attempt to generate APC in-situ. The synthetic genes encoding the functional domains of EPCR (1-193) and TM (224-462) with either a myc or flag linker along with a histidine tag were first assembled by PCA/PCR method, sequenced and the synthetic genes encoding the correct sequences for chimera I, chimera II, and TM2-His were successfully expressed in yeast Pichia host X-33 strain. Expressed protein was analyzed by Western blotting analysis, purified using Ni-chelate chromatography, and the ability of EPCR-TM chimera to catalyze PC activation was tested in solution phase. Overall, the expression level of chimera I, chimera II, and TM2-His was low, and mature proteins were partially secreted into the medium, and His-tag purification yielded partially pure products. Chimera containing EPCR and TM was noted to generate 1.8-fold higher APC compared to standard TM at comparable concentrations. iii DEDICATION To my husband Jacob and my daughter Cinae AUTHORS'S ACKNOWLEGEMENTS I would like to thank Dr. Subramanian for giving me an opportunity to work on this research project and for guidance and unlimited support to achieve my goal. whom provided their help for me to solve the problems. iv Table of Contents Chapter 1: Expression and characterization of chimera with anti-coagulant function Introduction 1 Materials and Methods 18 Results 37 Discussion 60 Conclusion 66 Future work 67 References 68 Appendices 74 v List of Table Table 1. List of sequence primers used for subcloned chimera I and chimera II into TOPO vector 21 Table 2. List of primers used for …