Transient expression of human prethrombin-2a in Nicotiana benthamiana.

ZALDÚA, Z; VELANDER W; WEIJIE X; ALVAREZ, MA; LAGUÍA BECHER, M; SATYANARAYANA T

Lima

Congreso; IX Encuentro REDBIO 2016; 2016

CIAT Perú

Human prothrombin is a complex vitamin K dependent glycoprotein involved in blood coagulation. Prothrombin is the zymogen to the enzyme thrombin which has been expressed in an active form in CHO cells. Plants (including in vitro cultures) is another possible vehicle for the production of recombinant proteins. Its advantages include rapid, cost effective scale-up and lack of contamination from specific mammalian pathogens. We have expressed in Nicotiana benthamiana prethrombin-2a (FII), the smallest and the post-translationally most simple of the zymogen precursors to thrombin. We have designed two constructs bearing the human FII coding sequence (864 bp), under the control of the CaMV35S promoter, fused to the signal peptide 2S2 from A. thaliana to deliver the protein to the secretory pathway, the Kozak sequence, and with or without adding the C-terminal KDEL sequence for retrieving the protein to the endoplasmic reticulum (pFII/-A/RE). Expression constructs consisting of the plant expression binary vectors pK7WG2 and p35SGAT (pK7WG2:pFII-A/RE and p35GAT:pFII-A/RE) were made and were introduced by electroporation into Agrobacterium tumefaciens. To analyze if post-transcriptional gene silencing (PTGS) could interfere with FII expression a co-agroinfiltration in the presence of Turnip crinkle virus capside protein (CP-TCV) was performed. Western blot analysis performed on plant extracts confirmed the protein integrity, showing a specific band of approximately 35 kDa in the two ER versions. Expression was highest in the PTGS CP-TCV suppressor, apoplastic versions. Our preliminary assessment shows that the recombinant pre-FII was expressed in the proper molecular weight range and structure needed for coagulation function.