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.

• Publication year

  1992

• Venue

  Journal of Biological Chemistry

• Publication date

  1992-04-25

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1016/s0021-9258(18)42393-9PMID 1569054
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product

  1991cited by this paper
• The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F.

  1991cited by this paper
• The E2F transcription factor is a cellular target for the RB protein.

  1991cited by this paper
• The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein.

  1991cited by this paper
• Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product.

  1991cited by this paper
• Tumor suppressor genes.

  1991cited by this paper
• Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor

  1991cited by this paper
• Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen.

  1990cited by this paper
• Expression of the human retinoblastoma gene product pp110RB in insect cells using the baculovirus system.

  1990cited by this paper
• The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations.

  1990cited by this paper
• Identification of HPV-16 E7 peptides that are potent antagonists of E7 binding to the retinoblastoma suppressor protein.

  1990cited by this paper
• Definition of the minimal simian virus 40 large T antigen- and adenovirus E1A-binding domain in the retinoblastoma gene product

  1990cited by this paper
• The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element.

  1989cited by this paper
• The retinoblastoma protein is phosphorylated during specific phases of the cell cycle.

  1989cited by this paper
• Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

  1989cited by this paper
• Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation.

  1989cited by this paper
• The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product.

  1989cited by this paper
• Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product

  1988cited by this paper
• Inactivation of the retinoblastoma susceptibility gene in human breast cancers.

  1988cited by this paper
• SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene.

  1988cited by this paper
• The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A.

  1988cited by this paper
• The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity

  1987cited by this paper
• Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication.

  1986cited by this paper
• Expression of recessive alleles by chromosomal mechanisms in retinoblastoma

  1983cited by this paper
• Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4

  1970cited by this paper

• Structural and functional analysis of cancer-associated missense variants in the retinoblastoma protein pocket domain

  2024cites this paper
• Differential regulation of E 2 F t r a n s-activation by cyclin / cdk 2 complexes

  2007cites this paper
• Biochemical and structural investigations of the retinoblastoma protein, its binding partners, and the BRG1 protein - a subunit of human SWI/SNF remodeling complexes

  2006cites this paper
• Biochemical and biophysical characterization of the retinoblastoma protein and its interacting partners

  2004cites this paper
• Destabilization of the Retinoblastoma Tumor Suppressor by Human Papillomavirus Type 16 E7 Is Not Sufficient To Overcome Cell Cycle Arrest in Human Keratinocytes

  2001cites this paper
• Coexpression of proteins in bacteria using T7-based expression plasmids: expression of heteromeric cell-cycle and transcriptional regulatory complexes.

  2000cites this paper
• Bacterial expression and spectroscopic characterization of soybean leghaemoglobin a.

  1998cites this paper
• Charge reversal of a critical active-site residue of cytochrome-c peroxidase: characterization of the Arg48-->Glu variant.

  1997cites this paper
• Rapid, efficient, large-scale purification of unfused, non-denatured E7 protein of cottontail rabbit papillomavirus.

  1996cites this paper
• Differential regulation of the synthesis and activity of the major cyclin‐dependent kinases, p34cdc2, p33cdk2, and p34cdk4, during cell cycle entry and progression in normal human T lymphocytes

  1995cites this paper
• Differential regulation of E2F transactivation by cyclin/cdk2 complexes.

  1994cites this paper
• Identification of a novel retinoblastoma gene product binding site on human papillomavirus type 16 E7 protein.

  1994cites this paper
• Retinoblastoma protein reverses DNA bending by transcription factor E2F.

  1994cites this paper
• Identification of discrete structural domains in the retinoblastoma protein. Amino-terminal domain is required for its oligomerization.

  1994cites this paper
• Translocation of retinoblastoma protein associated with tumor cell growth inhibition.

  1993cites this paper
• Transcription factor E2F binds DNA as a heterodimer.

  1993cites this paper
• Molecular characterization of the retinoblastoma susceptibility gene.

  1993cites this paper
• Comparison of the binding of the human papillomavirus type 16 and cottontail rabbit papillomavirus E7 proteins to the retinoblastoma gene product.

  1993cites this paper
• Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F

  1993cites this paper
• Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein

  1993cites this paper
• Papillomavirus E7 protein binding to the retinoblastoma protein is not required for viral induction of warts

  1993cites this paper
• Efficient purification of a full length and biochemically active p110Rb, the retinoblastoma gene product.

  1992cites this paper
• A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F.

  1992cites this paper