Recombinant enterokinase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities in fusion protein technology.
Enterokinase and recombinant enterokinase light chain (rEK(L)) 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 rEK(L) after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEK(L). Utilizing the secretion enhancer peptide derived from the human interleukin 1 beta, the recombinant EK(L) was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EK(L) was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EK(L), whereas the N-terminal His-tagged EK(L) was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EK(L) was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEK(L) 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.
Choi
S I
SI
Department of Biotechnology, College of Engineering and Bioproducts Research Center, Yonsei University, Seoul 120-749, Korea.
Song
H W
HW
Moon
J W
JW
Seong
B L
BL
D000345:Affinity Labels; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006868:Hydrolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000345:Affinity Labels; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D004765:Enteropeptidase