Selenolthiol and dithiol C-terminal tetrapeptide motifs for one-step purification and labeling of recombinant proteins produced in E. coli.
We have previously shown that a redox-active selenocysteine-containing tetrapeptide-Sel-tag (Gly-Cys-Sec-Gly)-can be used as a C-terminal fusion motif for recombinant proteins produced in Escherichia coli. This Sel-tag allows selenolate-targeted one-step purification, as well as fluorescent labeling or radiolabeling either with gamma emitters (75Se) or with positron-emitting radionuclides (11C). Here we have analyzed four different redox-active C-terminal motifs, carrying either dithiol (Gly-Cys-Cys-Gly or Ser-Cys-Cys-Ser) or selenolthiol (Gly-Cys-Sec-Gly or Ser-Cys-Sec-Ser) motifs. Utilizing these different functional motifs with the same recombinant protein (Fel d 1), we were able to assess their relative reactivities and potential usefulness for biotechnological applications. We found that all four redox-active tags could be utilized for efficient one-step purification to provide pure protein from a crude bacterial lysate through reversible binding to phenylarsine oxide sepharose, with yields and purities comparable to those obtained for a His-tagged protein purified by the more common approach with use of a Ni2+ column. For labeling with electrophilic fluorescent or radioactive compounds, however, the selenolthiol motifs were considerably more efficient than their dithiol counterparts. The results thus show that both the selenolthiol- and the dithiol-containing tags can serve as efficient alternatives to His-tags for protein purification, while the selenolthiol motifs offer additional and unique potential for Sec-targeted labeling. It should therefore be possible to utilize these multifunctional tetrapeptide motifs to develop a wide range of novel biotechnological applications based on Sec targeting with electrophilic compounds.
Cheng
Qing
Q
Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
Johansson
Linda
L
Thorell
Jan-Olov
JO
Fredriksson
Anna
A
Samén
Erik
E
Stone-Elander
Sharon
S
Arnér
Elias S J
ES
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D017279:Selenocysteine / Q000737:chemistry*
D005456:Fluorescent Dyes; D010455:Peptides; D011994:Recombinant Proteins; D017279:Selenocysteine