Transient expression and purification of prochymosin in tobacco (Nicotiana benthamiana) using viral vectors

Document Type : Research Paper

Authors

1 PhD Student, Department of Food Science and Technology, College of Agriculture, Shiraz University, Shiraz, Iran.

2 Department of Food Science and Technology, College of Agriculture, Shiraz University

3 Professor, Institute of Biotechnology, College of Agriculture, Shiraz University, Shiraz, Iran

4 Department of Food Science and Technology, College of Agriculture, Shiraz University Seafood Processing Research Group, College of Agriculture, Shiraz University

5 Department of Biochemistry, Shiraz University

Abstract

Objective
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. 
Material and Methods
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.
Results
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.
Conclusion
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.

Keywords

References

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Agricultural Biotechnology Journal
Volume 14, Issue 2
May 2022
Pages 45-66

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