Linked Life Data

8532685

Source:http://linkedlifedata.com/resource/pubmed/id/8532685

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
1996-2-1
pubmed:abstractText
The construction and characterization of a combinatorial library of a solvent-exposed surface of an alpha-helical domain derived from a bacterial receptor is described. Using a novel solid-phase approach, the library was assembled in a directed and successive manner utilizing single-stranded oligonucleotides containing multiple random substitutions for the variegated segments of the gene fragment. The simultaneous substitution of 13 residues to all 20 possible amino acids was carried out in a region spanning 81 nucleotides. The randomization was made in codons for amino acids that were modelled to be solvent accessible at a surface made up from two of the three alpha-helices of a monovalent Fc-binding domain of staphylococcal protein A. After cloning of the PCR-amplified library into a phagemid vector adapted for phage display of the mutants, DNA sequencing analysis suggested a random distribution of codons in the mutagenized positions. Four members of the library with multiple substitutions were produced in Escherichia coli as fusions to an albumin-binding affinity tag derived from streptococcal protein G. The fusion proteins were purified by human serum albumin affinity chromatography and subsequently characterized by SDS-electrophoresis, CD spectroscopy and biosensor analysis. The analyses showed that the mutant protein A derivatives could all be secreted as soluble full-length proteins. Furthermore, the CD analysis showed that all mutants, except one with a proline introduced into helix 2, have secondary structures in close agreement with the wild-type domain. These results proved that members of this alpha-helical receptor library with multiple substitutions in the solvent-exposed surface remain stable and soluble in E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0269-2139
pubmed:author
pubmed:issnType
Print
pubmed:volume
8
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
601-8
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
A combinatorial library of an alpha-helical bacterial receptor domain.
pubmed:affiliation
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't