Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli
Braz. arch. biol. technol
;
58(2): 154-165, Mar-Apr/2015. graf
Article
in English
| LILACS
| ID: lil-744315
ABSTRACT
Human enterokinase (synonym enteropeptidase, EC 3.4.21.9) light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia coli codon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 15000 (enzymesubstrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104 U/mg.
Full text:
Available
Index:
LILACS (Americas)
Language:
English
Journal:
Braz. arch. biol. technol
Journal subject:
Biology
Year:
2015
Type:
Article
/
Project document
Affiliation country:
China
Institution/Affiliation country:
Shanxi University/CN
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