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1.
J Biotechnol ; 340: 57-63, 2021 Nov 10.
Article in English | MEDLINE | ID: mdl-34506803

ABSTRACT

Enterokinase is one of the hydrolases that catalyze hydrolysis to regulate biological processes in intestinal visceral mucosa. Enterokinase plays an essential role in accelerating the process of protein digestion as it converts trypsinogen into active trypsin by accurately recognizing and cleaving a specific peptide sequence, (Asp)4-Lys. Due to its exceptional substrate specificity, enterokinase is widely used as a versatile molecular tool in various bioprocessing, especially in removing fusion tags from recombinant proteins. Despite its biotechnological importance, mass production of soluble enterokinase in bacteria still remains an unsolved challenge. Here, we present an effective production strategy of human enterokinase using tandemly linked solubility enhancers consisting of thioredoxin, phosphoglycerate kinase or maltose-binding protein. The resulting enterokinases exhibited significantly enhanced solubility and bacterial expression level while retaining enzymatic activity, which demonstrates that combinatorial design of fusion proteins has the potential to provide an efficient way to produce recombinant proteins in bacteria.


Subject(s)
Enteropeptidase , Escherichia coli , Amino Acid Sequence , Enteropeptidase/genetics , Enteropeptidase/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Recombinant Fusion Proteins/genetics , Recombinant Proteins/genetics , Solubility
2.
iScience ; 24(2): 102104, 2021 Feb 19.
Article in English | MEDLINE | ID: mdl-33615202

ABSTRACT

Systematic control of in vivo behavior of protein-based therapeutics is considered highly desirable for improving their clinical outcomes. Modulation of biochemical properties including molecular weight, surface charge, and binding affinity has thus been suggested to enhance their therapeutic effects. However, establishing a relationship between the binding affinity and tumor localization remains a debated issue. Here we investigate the influence of the binding affinity of proteins on tumor localization by using four repebodies having different affinities to EGFR. Biochemical analysis and molecular imaging provided direct evidence that optimal affinity with balanced target binding and dissociation can facilitate deep penetration and accumulation of protein binders in tumors by overcoming the binding-site-barrier effect. Our findings suggest that binding kinetics-based protein design can be implicated in the development of fine-tuned protein therapeutics for cancers.

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