Linker peptide and affinity tag for detection and purification of single-chain Fv fragments.
The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1. This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution. The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography. The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL). Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced. All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm. Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs.
Küttner
Gabriele
G
Institut für Biochemie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Germany. gabriele.kuettner@charite.de
Giessmann
Elke
E
Wessner
Helga
H
Scholz
Christa
C
Reinhardt
Dina
D
Winkler
Karsten
K
Marx
Uwe
U
Höhne
Wolfgang
W
D000345:Affinity Labels; D000818:Animals; D005591:Chemical Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000235:genetics*; D007128:Immunoglobulin Fragments / Q000032:analysis* / Q000235:genetics; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D010455:Peptides / Q000235:genetics*
D000345:Affinity Labels; D016655:HIV Core Protein p24; D007128:Immunoglobulin Fragments; D010455:Peptides; C055966:immunoglobulin Fv