Tools for the production and purification of full-length, N- or C-terminal 32P-labeled protein, applied to HIV-1 Gag and Rev.
We have constructed two new vectors for the production of foreign proteins in Escherichia coli. The vectors, pGEX-GTH and pET-HTG, produce protein fused to glutathione S-transferase (GST) at the N- and C-termini, respectively, allowing one-step purification on glutathione-Sepharose. Furthermore, they carry the recognition sequence (RRASV) for the catalytic subunit of cAMP-dependent heart muscle kinase (HMK) at the terminus distal to the GST tag, enabling specific 32P labeling in vitro. By positioning the GST and HMK sequences at opposite ends of the introduced gene, only full-length fusion protein becomes radiolabeled after purification. Avoiding the labeling of shorter fusion protein species, often observed in bacterial expression of foreign genes, is particularly important for a number of different purposes, including protein mobility shift analysis and protein footprinting technology.
Jensen
T H
TH
Department of Molecular Biology, University of Aarhus, Denmark.
Jensen
A
A
Kjems
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015683:Gene Products, gag / Q000235:genetics / Q000302:isolation & purification*; D015694:Gene Products, rev / Q000737:chemistry / Q000302:isolation & purification*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry*; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins / Q000302:isolation & purification*; D054321:rev Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015694:Gene Products, rev; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D005982:Glutathione Transferase