An efficient expression, purification and immunodetection system for recombinant gene products.
We describe a modification of the mammalian expression vector pRc/CMV, which drives expression of inserted genes from either the human cytomegalovirus (CMV) immediate-early promoter or the bacteriophage T7 RNA polymerase promoter. The modification is designed to allow expression, simple purification and specific immunodetection of recombinant fusion proteins. The modified plasmid, termed pTag/CMV-neo, encodes a Kozak consensus ribosome-binding site (RBS) and a 30-amino acid fusion tag peptide. This peptide consists of a metal ion-binding site, (His)6, for single-step affinity purification using Ni(2+)-chelating resin and a multi-purpose HIV-1-derived peptide (p18HIV). This viral epitope can be used to identify, detect and characterize target fusion proteins in conjunction with a specific monoclonal antibody H902 that does not display cross-reactivity with cellular proteins. The H902 production hybridoma cell line is reagent #521 from the NIH AIDS Research and Reference Program.
Baier
G
G
La Jolla Institute for Allergy and Immunology, CA.
Baier-Bitterlich
G
G
Couture
C
C
Telford
D
D
Giampa
L
L
Altman
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003587:Cytomegalovirus / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D014764:Viral Proteins
D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases