A new idea for simple and rapid monitoring of gene expression: requirement of nucleotide sequences encoding an N-terminal HA tag in the T7 promoter-driven expression in E. coli.
Mammalian expression vectors are used to overexpress genes of interest in mammalian cells. High temperature requirement protein A1 (HtrA1), used as a specific target, was expressed from the pHA-M-HtrA1 plasmid in HEK293T cells, inducing cell death. Expression of HtrA1 was driven by the pHA-M-HtrA1 mammalian expression vector in E. coli resulting in growth suppression of E. coli in an HtrA1 serine protease-dependent manner. By using various combinations of promoters, target genes and N-terminal tags, the T7 promoter and N-terminal HA tag in the mammalian expression vector were shown to be responsible for expression of target genes in E. coli. Thus the pHA-M-HtrA1 plasmid can be used as a novel, rapid pre-test system for expression and cytotoxicity of the specific target gene in E. coli before assessing its functions in mammalian cells.
Department of Medical Life Sciences, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea.
Jeong-Mi
JM
Moon
Goo-Young
GY
Kim
Hyangshuk
H
Rhim
D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D000074289:High-Temperature Requirement A Serine Peptidase 1; D006801:Humans; D015151:Immunoblotting; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000074289:High-Temperature Requirement A Serine Peptidase 1; C474012:HTRA1 protein, human; D012697:Serine Endopeptidases