Cloning and Expression of a Fibrin(ogen)olytic Serine Protease from Solanum tuberosum

WHITEHEART, SIDNEY W.; GUEVARA, M. GABRIELA; PEPE, ALFONSO

Memphis, TN

Conferencia; 16th Midwest Platelet Conference; 2016

The University of Tennessee

Introduction: Serine proteases from plants are proposed to have a number of clinical applications, including as potential anti-coagulants and anti-platelet agents. One such protease, StSBTc-3, Solanum tuberosum, was previously identified by our laboratory as a fibrinogenolytic enzyme. To further define its therapeutic value, we sought to generate recombinant StSBTc-3 for analysis. The StSBTc-3 gene was cloned from Solanum tuberosum DNA and recombinant protein was expressed in E. coli.Methods and Results: DNA was extracted from Solanum tuberosum tubers and the gene encoding StSBTc-3 (PGSC0003DMT400027148) was amplified by Polymerase Chain Reaction using gene-specific oligonucleotide primers. The fragment corresponding to the peptidase domain (Ala-61-Thr-504) was amplified, sequenced, and cloned into pPROEX HTb and pGEX-KG expression vectors. Rosetta E. coli cells were transformed and enzyme expression was induced with IPTG. The cells were harvested and the enzyme was purified. Low levels of soluble StSBTc-3 activity were obtained. However, a larger amount of insoluble enzyme was generated. Rapid refolding and slower dialysis techniques failed to generate soluble, active enzyme. Further testing of bacterial and eukaryotic expression systems is underway to generate soluble recombinant enzyme that retains activity.Conclusions: After successfully cloning the DNA encoding the peptidase domain of StSBTc-3 from Solanum tuberosum, we can express the intact full-length domain; however, the recombinant protein produced in E. coli is insoluble. Other expression systems (e.g. Rosetta-gami 2 host strains) are being explored to generate active StSBTc-3 for study in hemostasis assays.