Antiferritin single-chain antibody: a functional protein with incomplete folding?
The pET(scF11) plasmid was constructed comprising the gene of a single-chain antibody against human ferritin. This plasmid encodes the leader peptide pelB followed by the heavy chain variable V(H) domain, (Gly4Ser)3 linker peptide, and light chain variable V(L) domain. The correctly processed scF11 antibody was expressed in Escherichia coli as an insoluble protein without the leader peptide. Purified soluble scF11 was obtained after solubilization in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion-exchange chromatography. ScF11 demonstrated only a approximately 8-fold decrease in the affinity (Ka = 5.1 x 10(8) M(-1) in RIA and 1.8 x 10(8) M(-1) in ELISA) vs. the parent IgG2a/kappa monoclonal antibody F11. The emission maximum of intrinsic fluorescence strongly suggests a compact conformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein. However, scF11 demonstrated (i) the lack of denaturant-induced fluorescence 'dequenching' effect characteristic of the completely folded parent antibody, and (ii) prominent binding, under physiological conditions, of a hydrophobic probe 8-anilino-1-naphthalenesulfonate (ANS) recognizing partially structured states of a protein. These findings are indicative of an incomplete tertiary fold that gives ANS access to the protein hydrophobic core. This work provides the first indication that the functional single-chain antibody scF11 displays some properties of a partially structured state and therefore may possess incomplete folding.
Martsev
S P
SP
Institute of Bio-Organic Chemistry, National Academy of Sciences of Belarus, Minsk. martsev@ns.iboch.ac.by
Kravchuk
Z I
ZI
Chumanevich
A A
AA
Vlasov
A P
AP
Dubnovitsky
A P
AP
Bespalov
I A
IA
Arosio
P
P
Deyev
S M
SM
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005293:Ferritins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D017931:DNA Primers; D011994:Recombinant Proteins; C116856:scF11 antibody; D005293:Ferritins