[Screening of highly active recombinant bovine enterokinase mutants mediated by green fluorescent protein and its soluble expression in Escherichia coli].

Authors

Changchun Han, Junhao Yue, Yuwen Tu, Zhuhua Chan, Lehe Mei, Changjiang Lyu

Published in

Sheng wu gong cheng xue bao = Chinese journal of biotechnology. Volume 42. Issue 9. Pages 4038-4051. Sep 25, 2026.

Abstract

Bovine enterokinase light chain (EK_L), a serine protease that specifically recognizes the Asp-Asp-Asp-Asp-Lys (DDDDK) sequence, is widely used for the specific cleavage of affinity tags in recombinant proteins. However, EK_L contains multiple cysteine residues and four disulfide bonds, making it difficult to fold correctly, and it tends to form inactive inclusion bodies when expressed in Escherichia coli. To achieve efficient soluble expression of EK_L in E. coli and screen high-activity mutants, co-expression of the DnaK-DnaJ-GrpE and GroES-GroEL molecular chaperones was adopted to enhance its solubility. Subsequently, the DDDDK sequence was site-specifically introduced into the Loop region of superfolder green fluorescent protein (sfGFP) to generate the fluorescent substrate sfGFP^M for EK_L. Based on the principle of fluorescence loss, a high-throughput screening method for highly active EK_L variants was established. Using error-prone PCR, a random mutagenesis library of the EK_L gene was constructed, from which the EK_L-S42M/I139D mutant was obtained. This mutant showed a 2.56-fold increase in catalytic efficiency and significantly improved thermostability. Molecular dynamics simulations revealed that the S42M/I139D mutations synergistically optimized the stability and activity of EK_Lvia distal allosteric regulation. Meanwhile, fed-batch fermentation further enhanced the soluble expression level of EK_L-S42M/I139D, with a yield of 203 mg/L and an enzymatic activity of 7.45×10⁴ U/mL. This study not only achieves the soluble expression of EK_L in E. coli and obtains a highly active mutant, but also provides a visual tool for the rapid detection of EK_L activity.

PMID:
42773660
Bibliographic data and abstract were imported from PubMed on 23 Sep 2026.

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