Linked Life Data

1967024

Source:http://linkedlifedata.com/resource/pubmed/id/1967024

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1992-8-26
pubmed:abstractText
A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
1
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
169-76
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1990
pubmed:articleTitle
Expression of mammalian liver glycolytic/gluconeogenic enzymes in Escherichia coli: recovery of active enzyme is strain and temperature dependent.
pubmed:affiliation
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794-8661.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.