Purification and characterization of a functionally homogeneous 60-kDa species of the retinoblastoma gene product.
The retinoblastoma susceptibility gene (RB) encodes a 928-amino acid protein (pRB) that is hypothesized to function in a pathway that restricts cell proliferation. The immortalizing proteins from three distinct DNA tumor viruses (SV40 large T antigen, adenovirus E1a, and human papilloma virus Type 16 E7) have been shown to interact with RB protein through two noncontiguous regions comprised of amino acids 393-572 (domain A) and 646-772 (domain B). We constructed a truncated form of RB (RB p60) that retains these two domains but eliminates the N-terminal 386 amino acids of RB. RB p60 was expressed in Escherichia coli in inclusion bodies. After solubilization, it was refolded in the presence of magnesium chloride, and the active protein was isolated with an E7 peptide affinity column. The protein that elutes from this column is functionally homogenous in its ability to bind immobilized E7 protein. Thermal denaturation studies provide additional evidence for the conformational homogeneity of the isolated protein. This purification scheme allows the isolation of significant amounts of RB p60 protein that is suitable for structural and functional studies.
Edwards
G M
GM
Department of Cancer Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Huber
H E
HE
DeFeo-Jones
D
D
Vuocolo
G
G
Goodhart
P J
PJ
Maigetter
R Z
RZ
Sanyal
G
G
Oliff
A
A
Heimbrook
D C
DC
D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006358:Hot Temperature; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D050725:Papillomavirus E7 Proteins; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D016160:Retinoblastoma Protein / Q000302:isolation & purification* / Q000378:metabolism
D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016160:Retinoblastoma Protein; C059731:oncogene protein E7, Human papillomavirus type 16; D004247:DNA