Shahid Bahonar University of Kerman and Iranian Biotechnology Society Agricultural Biotechnology Journal 2228-6705 14 2 2022 06 22 Transient expression and purification of prochymosin in tobacco (Nicotiana benthamiana) using viral vectors Transient expression and purification of prochymosin in tobacco (Nicotiana benthamiana) using viral vectors 45 66 3287 10.22103/jab.2021.16588.1262 FA Esmaeil Ziaee PhD Student, Department of Food Science and Technology, College of Agriculture, Shiraz University, Shiraz, Iran. Mohammad Hadi Eskandari Department of Food Science and Technology, College of Agriculture, Shiraz University Ali Niazi Professor, Institute of Biotechnology, College of Agriculture, Shiraz University, Shiraz, Iran Marzieh Moosavi Nasab Department of Food Science and Technology, College of Agriculture, Shiraz University Seafood Processing Research Group, College of Agriculture, Shiraz University Mahmmod Aminlari Department of Biochemistry, Shiraz University Journal Article 2021 01 08 Objective<br />The aim of present study was transient expression and purification of recombinant caprine chymosin and prochymosin in N. benthamiana leaves using plant binary vectors and tobacco mosaic virus (TMV) vector. <br />Material and Methods<br />The caprine prochymosin gene sequence was obtained from NCBI gene bank and optimized at codons for preferential expression in tobacco leaves. The chymosin and prochymosin genes were introduced into the plant binary and viral vectors.  Immunoblotting analyses were conducted to demonstrate expression of chymosin and prochymosin, and the expression level was quantified by milk clotting activity test.<br />Results<br />Results indicated that N. benthamiana leave cells cannot process prochymosin correctly and only a weak band observed in extracted total proteins (TPs) which showed no milk clotting activity while leaves infiltrated by p20αchymosin showed a band with chymosin molecular weight and slightly showed milk clotting activity. TMV-based recombinant prochymosin and chymosin expression showed necrosis after 3 days post infiltration (dpi) in agro-infiltrated leaves and after 5 days, leaves completely dried and necrotized. Blotting analysis showed only a band with right molecular weight in TSPs extracted N. benthamiana leaves infiltrated by TMVαchymosin.  Moreover, we subcloned chymosin which expanded with a N-terminal barley alpha amylase signal peptide and a C-terminal hybrid Fc tag which labelled as TMVαchymosinhyFc. The expression level increased gradually from 2 to 5 days after infiltration from 3.3 U/g FW to 16.8 U/g FW but after 5-day leaves necrosis started and expression level reduced gradually.<br />Conclusion<br />Viral vectors and transient expression can be a cheap, fast and effective method to produce a huge amount of recombinant proteins but it seems chymosin production still needs more research and find the N. benthamiana cells problem with chymosin which leads to necrosis and reduce the expression level for large scale production. Objective<br />The aim of present study was transient expression and purification of recombinant caprine chymosin and prochymosin in N. benthamiana leaves using plant binary vectors and tobacco mosaic virus (TMV) vector. <br />Material and Methods<br />The caprine prochymosin gene sequence was obtained from NCBI gene bank and optimized at codons for preferential expression in tobacco leaves. The chymosin and prochymosin genes were introduced into the plant binary and viral vectors.  Immunoblotting analyses were conducted to demonstrate expression of chymosin and prochymosin, and the expression level was quantified by milk clotting activity test.<br />Results<br />Results indicated that N. benthamiana leave cells cannot process prochymosin correctly and only a weak band observed in extracted total proteins (TPs) which showed no milk clotting activity while leaves infiltrated by p20αchymosin showed a band with chymosin molecular weight and slightly showed milk clotting activity. TMV-based recombinant prochymosin and chymosin expression showed necrosis after 3 days post infiltration (dpi) in agro-infiltrated leaves and after 5 days, leaves completely dried and necrotized. Blotting analysis showed only a band with right molecular weight in TSPs extracted N. benthamiana leaves infiltrated by TMVαchymosin.  Moreover, we subcloned chymosin which expanded with a N-terminal barley alpha amylase signal peptide and a C-terminal hybrid Fc tag which labelled as TMVαchymosinhyFc. The expression level increased gradually from 2 to 5 days after infiltration from 3.3 U/g FW to 16.8 U/g FW but after 5-day leaves necrosis started and expression level reduced gradually.<br />Conclusion<br />Viral vectors and transient expression can be a cheap, fast and effective method to produce a huge amount of recombinant proteins but it seems chymosin production still needs more research and find the N. benthamiana cells problem with chymosin which leads to necrosis and reduce the expression level for large scale production. https://jab.uk.ac.ir/article_3287_b46bef4f95c6e5bf299491c6b496ec7d.pdf