Recombinant expression of bovine enteropeptidase light chain in SHuffle® T7 Express as a new host and optimization of induction parameters
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mohammad shoae,1 Hossein Safarpour ,2 Mohsen Khorashadizadeh,3,*
1. Department of medical Biotechnology, faculty of medicine, Birjand University of Medical Sciences
2. Department of medical Biotechnology, faculty of medicine, Birjand University of Medical Sciences
3. Department of medical Biotechnology, faculty of medicine, Birjand University of Medical Sciences
- Introduction: Enteropeptidase is an enzyme consists of two subunits including heavy chain and light chain. The light chain of enteropeptidase (EPL) is the proteolytic subunit that specifically recognizes (Asp)4-Lys amino acid sequence and cuts after lysine amino acid. Due to the importance of this enzyme in laboratory and industry for purification of expressed protein in fusion with a fusion tag, a lot of study has performed on the EPL expression using different prokaryotic and eukaryotic expression hosts including yeasts and Escherichia coli. SHuffle® T7 Express is an engineered strain of E. coli that is valuable for the expression of proteins that contain disulfide bonds to form the protein correctly. It is also capable of refolding mis-oxidized bonds to form in a correct way. Enteropeptidase light chain contains multiple disulfide bonds in its structure and it is indicated that it is a hard to express protein. In this research, we applied the SHuffle® T7 strain and we evaluated the potential of this strain as a new host to express and correct folding of EPL. We subcloned the DNA encoding EPL into pET-32b plasmid in frame with thioredoxin tag that is a solubilization enhancing factor. Then, we evaluated the proteolytic activity of purified EPL and expression condition optimized using response surface methodology (RSM).
- Methods: Vector and bacterial culture media:
Subcloning was performed by restriction cloning. Amplifying the EPL DNA sequence was done by PCR using pET-28a plasmid containing the DNA sequence of EPL as template. The amplified sequence was then inserted into pET-32b.
Chemical transformation and expression:
Standard protocol of Chemical method was applied to prepare competent cells. After overnight cultivation of a transformed colony, one ml of the cultivation was added to fresh medium in a volume of 100 mL containing 100 µg mL-1 ampicillin and incubated at 37 °C in a shaker incubate with the speed of 160 rpm to reach the OD600 of 0.8. Then, 1 mM IPTG was inoculated to induce protein expression and incubation was continued for four h.
Extraction of expressed protein and purification:
After extraction by lysis buffer containing 1 mgr/ml lysozeme, Urea gradient was exerted to solubilize and recovery of protein structure from inclusion bodies. Then, Ni-NTA purification system was used to purify the protein.
Biological assay:
Using different value and ratio of the EPL to its substrate (Thioredoxin/hGH fusion protein), the activity of EPL was assayed and incubation of reaction tubes was done at 37 °C for 12 h.
Parameters optimization using RSM optimization approach:
Central composite design (CCD) was used to optimize the significant factors for protein expression including temperature, the concentration of inducer, incubation time and OD of induction.
- Results: Cloning of EPL DNA in pET-32b:
EPL DNA was amplified from pET-28a by PCR and cloned correctly into pET-32b by restriction cloning.
Expression and purification:
Primary analyze was indicated that inclusion bodies were formed. Solubilization of inclusion bodies was performed by urea gradient method. The concentration of purified protein was 200 microgram/ml.
Biological assay:
The results indicated that the substrate was digested by proteolytic activity of EPL into two protein fragments thioredoxin and hGH.
Optimization of enteropeptidase production:
Optimized parameters were 1.05 mM inducer, OD of induction 0.6, seven hour incubation after induction, and 26.5 °C for temperature. The yield of expression in this condition was 1780.85 µg mL-1 that is a high level of expression.
- Conclusion: In this research, expression and RSM optimization of EPL was conducted and the active enzyme was achieved. Before optimization, a comparable expression value with previous researches was achieved. Optimization resulted in the high level of protein expression (1780.85 µg mL-1) in the condition of 1.05 mM inducer (IPTG) at OD: 0.6 and seven h incubation at 26.5 °C for EPL. However, using SHuffle® T7 Express as a capable strain in correct folding of protein was not effective on the cytoplasmic expression in soluble form.
- Keywords: Enteropeptidase light chain, SHuffle T7 Express, parameter optimization.
