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Confining human tissue transglutaminase (htTG) into plant secretory pathway as strategy to increase protein yields
Congreso; Plant Based Vaccines, Antibodies and Biologics PBVA 2013; 2013
Human tissue transglutaminase (htTG) plays an important role in celiac disease (CD) pathogenesis and htTG-specific immunoglobulin G and A in human sera are useful diagnostic markers. Screening test for CD requires of good quality htTG antigen to increase the efficacy of the serological assay. Transglutaminases (TG) are a group of enzymes characterized by protein cross-linking activity what results in toxic effects on cell developing and growing. In addition TG has autoproteolytic activity what also decrease recombinant protein yields. In consequence, htTG is a very difficult protein to be produced in both prokaryotic and eukaryotic expression systems. In this work, htTG was introduced into plant secretory pathway as strategy to reduce its toxic effects on cellular metabolism and to increase protein stability. To this end, htTG was fused to an eukaryotic signal peptide. To avoid secretion, the endoplasmic reticulum (ER) retention signal SEKDEL and the C-terminal vacuolar sorting signal KISIA (CtVSS) were also introduced at the C-terminus to produce ER-htTG and vac-htTG, respectively. As controls, cytoplasmic (cyt-htTG) and secretory (sec-htTG) versions were also obtained. To facilitate purification a six histidine tag was also fused to htTG. The four htTG constructs were introduced in Agrobacterium tumefaciens GV3101 and leaves of Nicotiana benthamiana were infiltrated with these agrobacteria. Six days after infiltration, leaves were collected and htTG was purified using His Spin Trap (GE) affinity chromatography. Bound htTG was eluted at pH 4.0 and eluted fractions were analyzed by immunoblot with three htTG-specific monoclonal antibodies (mAb) that recognize different epitopes (2G3, 5G7, 4E1) and a His-tag specific polyclonal serum. Cyt-htTG and sec-htTG were not detected while both ER-htTG and vac-htTG were recognized by the three antibodies. Protein yields were 3.1 and 1,9 ug /g of vac-htTG and ER-htTG respectively. When a construct encoding SP-Cherry-htTG-CtVSS was infiltrated, red fluorescence was observed in the central vacuole and the full-length Cherry-htTG-CtVSS protein was detected by inmunoblot analysis confirming the stability of this fusion on the vacuole. Finally, an ELISA was performed with sera of CD patients and healthy controls. Both vac-htTG and ER-htTG were positively recognized by IgA of CD patients confirming the effectiveness of these plant produced htTG versions. In conclusion, this work shows that vacuolar htTG accumulates in the central vacuole of leaves and that both vac-htTG and ER-htTG are stable and can be easily purified from leaves. Importantly the two plant purified versions of htTG are recognized by htTG-specifc mAbs and sera of CD patients while are not recognized by non celiac sera. These results point out that plant vac-htTG and ER-htTG purified antigens can be useful to develop assays for CD screening test.