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 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.
Kelley
K C
KC
Procept, Inc., Cambridge, MA 02139, USA.
Huestis
K J
KJ
Austen
D A
DA
Sanderson
C T
CT
Donoghue
M A
MA
Stickel
S K
SK
Kawasaki
E S
ES
Osburne
M S
MS
D017123:Bacteriophage T7 / Q000235:genetics*; D015704:CD4 Antigens / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007544:Isopropyl Thiogalactoside; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D015704:CD4 Antigens; D011994:Recombinant Proteins; C076613:recombinant soluble CD4; D007544:Isopropyl Thiogalactoside