Linked Life Data

9891990

Source:http://linkedlifedata.com/resource/pubmed/id/9891990

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1999-2-5
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0014-5793
pubmed:author
pubmed:issnType
Print
pubmed:day
28
pubmed:volume
441
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
458-62
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
Antiferritin single-chain antibody: a functional protein with incomplete folding?
pubmed:affiliation
Institute of Bio-Organic Chemistry, National Academy of Sciences of Belarus, Minsk. martsev@ns.iboch.ac.by
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't