Regulation of sCD4-183 gene expression from phage-T7-based vectors in Escherichia coli

Regulation of sCD4-183 gene expression from phage-T7-based vectors in Escherichia coli

In this paper, we describe various parameters affecting the regulation of expression of the sCD4-183 gene, encoding the 183-amino-acid soluble human two-domain CD4 protein, from phage-T7-based pET vectors. We demonstrated that for the sCD4-183 protein, the highest protein yield was obtained using ve...

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Bibliographic Details
Published in:Gene Vol. 156; no. 1; pp. 33 - 36
Main Authors: Kelley, Kevin C., Huestis, Kevin J., Austen, Douglas A., Sanderson, Christine T., Donoghue, Mary A., Stickel, Shawn K., Kawasaki, Ernest S., Osburne, Marcia S.
Format: Journal Article
Language:English
Published: Netherlands Elsevier B.V 14-04-1995
Subjects:
Bacteriophage T7 - genetics
CD4
CD4 Antigens - biosynthesis
CD4 Antigens - genetics
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
expression vector
Gene Expression Regulation
Genetic Vectors - genetics
Genetic Vectors - metabolism
Humans
IPTG induction
Isopropyl Thiogalactoside
phage T7
Plasmid stability
promoter
protein production
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Ab
Km
Plasmid stability
E
nt
Cm
bp
Ap
Tc
IPTG induction
T
bla
cat
kb
kan
promoter
expression vector
sCD4-183
Nm
aa
SDS
PAGE
T7p
®
CD4
lacO
IPTG
PCR
protein production
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Summary:In this paper, we describe various parameters affecting the regulation of expression of the sCD4-183 gene, encoding the 183-amino-acid soluble human two-domain CD4 protein, from phage-T7-based pET vectors. We demonstrated that for the sCD4-183 protein, the highest protein yield was obtained using vector pET-9a, in which neither expression of the T7 RNA polymerase-encoding gene nor the target gene was tightly regulated. The highest overall protein yield was obtained from cells grown for 24 h in the absence of inducer, a strategy that may be generally useful for production of less toxic proteins. We also describe two modifications of the pET vector system that effectively minimized leaky (uninduced) expression and enhanced plasmid stability. These have potential use in the production of toxic proteins, or of non-toxic proteins produced in high-density cultures.
Bibliography:ObjectType-Article-2
SourceType-Scholarly Journals-1
ObjectType-Feature-1
content type line 23
ISSN:0378-1119
1879-0038
DOI:10.1016/0378-1119(95)00008-T