Linked Life Data

2188684

Source:http://linkedlifedata.com/resource/pubmed/id/2188684

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1990-7-2
pubmed:abstractText
The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5' and 3' regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3. The 5' end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5' region. This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide. Variable amounts of the 3' end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III. The resulting blunt-ended 3' fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon. The modified 5' and 3' ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3. In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea - sodium dodecyl sulphate - polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures. Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-beta-thiogalactoside. Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E. coli, but the amounts were very small.(ABSTRACT TRUNCATED AT 250 WORDS)
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0829-8211
pubmed:author
pubmed:issnType
Print
pubmed:volume
68
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
559-66
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1990
pubmed:articleTitle
Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs.
pubmed:affiliation
Department of Obstetrics and Gynaecology, University of Western Ontario, London, Canada.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't