Recombinant enterokinase light chain with affinity tag: Expression from Saccharomyces cerevisiae and its utilities in fusion protein technology

Seong Il Choi, Hye Won Song, Jae Woong Moon, Baik L. Seong

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Enterokinase and recombinant enterokinase light chain (rEKL) have been used widely to cleave fusion proteins with the target sequence of (Asp)4-Lys. In this work, we show that their utility as a site-specific cleavage agent is compromised by sporadic cleavage at other sites, albeit at low levels. Further degradation of the fusion protein in cleavage reaction is due to an intrinsic broad specificity of the enzyme rather than to the presence of contaminating proteases. To offer facilitated purification from fermentation broth and efficient removal of rEKL after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEKL. Utilizing the secretion enhancer peptide derived from the human interleukin 1β, the recombinant EKL was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EKL was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EKL, whereas the N-terminal His-tagged EKL was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EKL was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEKL extremely useful for purification, post-cleavage removal, recovery, and recycling and will broaden the utility and the versatility of the enterokinase for the production of recombinant proteins.

Original languageEnglish
Pages (from-to)718-724
Number of pages7
JournalBiotechnology and Bioengineering
Volume75
Issue number6
DOIs
Publication statusPublished - 2001 Dec 20

Fingerprint

Enteropeptidase
Yeast
Saccharomyces cerevisiae
Fusion reactions
Technology
Proteins
Light
Purification
Nickel
Affinity chromatography
Recombinant proteins
Fermentation
Peptides
Recycling
Enzymes
Protein C
Interleukin-1
Affinity Chromatography
Recombinant Proteins
Histidine

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

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abstract = "Enterokinase and recombinant enterokinase light chain (rEKL) have been used widely to cleave fusion proteins with the target sequence of (Asp)4-Lys. In this work, we show that their utility as a site-specific cleavage agent is compromised by sporadic cleavage at other sites, albeit at low levels. Further degradation of the fusion protein in cleavage reaction is due to an intrinsic broad specificity of the enzyme rather than to the presence of contaminating proteases. To offer facilitated purification from fermentation broth and efficient removal of rEKL after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEKL. Utilizing the secretion enhancer peptide derived from the human interleukin 1β, the recombinant EKL was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EKL was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EKL, whereas the N-terminal His-tagged EKL was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EKL was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEKL extremely useful for purification, post-cleavage removal, recovery, and recycling and will broaden the utility and the versatility of the enterokinase for the production of recombinant proteins.",
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Recombinant enterokinase light chain with affinity tag : Expression from Saccharomyces cerevisiae and its utilities in fusion protein technology. / Choi, Seong Il; Song, Hye Won; Moon, Jae Woong; Seong, Baik L.

In: Biotechnology and Bioengineering, Vol. 75, No. 6, 20.12.2001, p. 718-724.

Research output: Contribution to journalArticle

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