Linked Life Data

7538852

Source:http://linkedlifedata.com/resource/pubmed/id/7538852

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1995-6-26
pubmed:abstractText
HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-l fermentor with 14-l net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-beta-D-thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS-PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
1572-6495
pubmed:author
pubmed:issnType
Print
pubmed:day
3
pubmed:volume
664
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
107-18
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Laboratory-scale production and purification of recombinant HIV-1 reverse transcriptase.
pubmed:affiliation
Institute of Applied Microbiology, Universität für Bodenkultur, Vienna, Austria.
pubmed:publicationType
Journal Article