Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening.
Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.
Sydor
Jens R
JR
Zyomyx, Inc., 26101 Research Road, Hayward, California 94545, USA.
Mariano
Maria
M
Sideris
Steve
S
Nock
Steffen
S
D000906:Antibodies / Q000737:chemistry; D000941:Antigens / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure; D005456:Fluorescent Dyes; D007118:Immunoassay / Q000379:methods; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D002479:Inclusion Bodies / Q000235:genetics; D015335:Molecular Probes / Q000096:biosynthesis / Q000138:chemical synthesis*; D019912:Mycobacterium xenopi / Q000235:genetics; D011489:Protein Denaturation; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis; D012235:Rhodamines
D000906:Antibodies; D000941:Antigens; D005456:Fluorescent Dyes; D007135:Immunoglobulin Variable Region; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D012235:Rhodamines