Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli

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 1:5000 (enzyme:substrate). 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.

The flexibility of the non-conservative region at the C terminus of D-hydantoinase from Pseudomonas putida YZ-26 is extremely limited.

We previously reported that a deletion mutant (P478) with a residue Arg deleted at the C terminus of D-hydantoinase (P479) from Pseudomonas putida YZ-26 was dissociated into the monomer from its dimeric state. Based on the above result, a series of mutants of the enzyme with the C-terminal residues either deleted or substituted were prepared. The size-exclusion chromatography and bioactivity assay show that a C-terminal-substituted enzyme (R479D) and several truncated mutants (P478, P477, P476, and P475) are dissociated into the monomeric state as well, but their activities are largely retained. In contrast, two other mutants (R474 and R479A) are expressed in the form of random aggregates without any activity. Our experiments demonstrate that only the last four amino acids (-PVQR) at the C terminus of the enzyme can be deleted without seriously affecting its activity, although the enzyme is dissociated from a dimer into a monomer. These mutants also reveal some unique properties such as the enzymatic activity in vivo or in vitro, the effect of divalent metal ions, and the thermostability etc. in comparison to wild-type enzyme (P479). In addition, the three-dimensional structural modeling shows that the intact structure of the enzyme is essential, and the flexibility of the non-conservative region at the C terminus of the enzyme is quite limited.

[Molecular form and stability of D-hydantoinase deleted at C-terminal residue Arg].

This report is about only deleting one C-terminal residue of D-hydantoinase to result in obvious changes on its molecular form and stability. A recombinant D-hydantoinase (P479) and its mutant enzyme deleted at C-terminal residue Arg (P478) were prepared by methods of gene cloning, expression and purification. Results show that the subunit molecular weight of P479 and P478 is the same (54kDa) as determined by SDS-PAGE, whilst the molecular form of native P479 and P478 is a dimer and a monomer respectively in the completely operative conditions. Compared with P479, the enzymatic activity of P478 for substrate hydantoin maintained about 40% and pH stability was obviously increased, at the alkaline side in particular, as well as the anti-SDS ability was also raised. However, the thermal stability for P478 was clearly lowed as compared to P479. It implies from above data that the C-terminal residue Arg of the D-hydantoinase is a crucial one for subunit dissociation, but non-essential for catalysis.