Expression feasibility of recombinant enterokinase in nicotiana benthamiana

Abstract

Enterokinase (EK) is the main enzyme used to cleave proteins that are produced in fusion format in the expression host. Recombinant bovine and human EK are widely used in cleavage of fusion proteins, because natural enzymes isolated from bovine intestines is prone to gut protease contamination and thus has ethical functional implications. The limiting factor in fusion protein technology is the isolation of the target protein from fusion protein. Enterokinase is predominantly used because of its unique cleavage pattern that cleaves without leaving N-terminal residues on the target protein. Owing to its attractive cleavage pattern and its versatility, EK is frequently used in laboratories. The demand for EK as an industrial reagent is high. Recombinant EK has been expressed and purified from various production systems but the yields are not high enough to meet the growing demand, resulting in high prices of recombinant EK, increasing costs of protein processing. Enterokinase is currently produced in Escherichia coli, Chinese hamster ovary (CHO) and yeast cells, but at very low yields resulting in limited availability. Enterokinase as a cleavage reagent remains active at various temperatures, wide pH range and in the presence of detergents and denaturants. These properties make it widely used in process which liberate proteins of interest from fusion constructs; which is the limiting step in recombinant protein yield. It is therefore worthwhile to explore other technologies to produce this high value protein and thus make it more affordable EK to researchers. The current study explored the feasibility of producing recombinant EK in Nicotiana benthamiana as an alternative to the current production systems. Two research questions that were investigated were the role of sub-cellular targeting in the observed level of expression and accumulation of the target protein, as well as the functionality or otherwise of the protein produced in different organelles. Bovine EK light chain gene was cloned into tobacco mosaic virus (TMV)-deconstructed vector and expressed via Agrobacterium mediated transfer. Leaves of N. benthamiana were transfected by vacuum infiltration to deliver the transformed Agrobacterium into the leaves for transient expression targeted to the apoplast and the cytosol. Plant-made EK was isolated from the plant matrix and biochemically analysed by SDS-PAGE gel, N-terminal peptide sequencing and western blot. EK was successfully visible as a protein band on an SDS-PAGE gel. The protein band was isolated and the sequence was confirmed by N terminal sequencing as well as western blot analysis. Using an anti-EK monoclonal antibody plant made EK was quantified by ELISA, and the expression amounts were established at 40 μg/g fresh weight when targeted to the apoplast. Cytosolic and apoplastic plant-made EK were partially purified and tested for kinetic activity. The activity assay measured cleavage of a thioester, ZLysSBZL by plant-made EK. Plant-made EK was shown to be active at 23 U/ml plant extract. Apoplast targeted EK had higher expression and was more functionally active compared to cytosol targeted EK. In conclusion, the current study demonstrated that the active bovine EK light chain could be expressed in leaves of N. benthamiana at detectable amounts making plants a genuine contender for making recombinant EK.