Linked Life Data

16269745

Source:http://linkedlifedata.com/resource/pubmed/id/16269745

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
2005-11-4
pubmed:abstractText
The strong LAC4 promoter (P(LAC4)) from Kluyveromyces lactis has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of P(LAC4) to promote gene expression in Escherichia coli. This can interfere with the process of assembling expression constructs in E. coli cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of P(LAC4) variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the E. coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with P(LAC4). Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by approximately 87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in K. lactis cells to levels comparable to wild-type P(LAC4). We created a novel integrative expression vector (pKLAC1) containing the P(LAC4) variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in E. coli cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in E. coli prior to their expression in K. lactis.
pubmed:commentsCorrections
http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-100226, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-11210145, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-1326415, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-1398092, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-15040944, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-1748298, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-2546684, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-6800877, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-6810093, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-8092859, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-8920185, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-9251219, http://linkedlifedata.com/resource/pubmed/commentcorrection/16269745-9872759
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0099-2240
pubmed:author
pubmed:issnType
Print
pubmed:volume
71
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7092-8
pubmed:dateRevised
2009-11-18
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in K. lactis.
pubmed:affiliation
New England Biolabs, 240 County Road, Ipswich, MA 01938-2723, USA.
pubmed:publicationType
Journal Article