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Identification, purification, and subcellular localization of prostate-specific membrane antigen PSM' protein in the
L S Grauer1, K D Lawler, J L Marignac
1Hybritech Incorporated, Beckman Coulter, Inc., San Diego, California 92196-9006, USA.
Abstract:
An alternatively spliced variant of prostate-specific membrane antigen (PSMA) designated PSM' was originally described following identification of its mRNA in normal prostate. We have purified the PSM' protein from LNCaP cells using two immunoaffinity columns in tandem. The first column contained a monoclonal antibody (7E11) that was reactive with the NH2 terminus of PSMA, which specifically depleted the LNCaP lysate of full-length PSMA. The nonbinding fraction was then passed over a second column composed of a monoclonal antibody (PEQ226.5), the epitope of which was located within the 134-437 domain of PSMA and shared with PSM'. The protein eluted from the second immunoaffinity column produced a Mr 95,000 band on SDS-PAGE, which was slightly lower than the full-length PSMA at Mr 100,000. The band was NH2-terminally sequenced through 15 residues, and the assigned sequence coincided with the predicted sequence for PSM' protein minus the first two NH2 terminus amino acids. The PSM' protein, therefore, began with residue 60 of PSMA (alanine). LNCaP cells were fractionated, and PSM' was localized to the cytoplasm.
Insights
Researchers purified the prostate-specific membrane antigen (PSMA) splice variant, PSM
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Prostate-specific membrane antigen (PSMA) is a transmembrane protein implicated in prostate cancer.
- An alternatively spliced variant, PSM', has been identified at the mRNA level in normal prostate tissue.
Purpose of the Study:
- To purify and characterize the PSM' protein from LNCaP cells.
- To determine the N-terminus and cellular localization of the PSM' protein.
Main Methods:
- Immunoaffinity chromatography using two monoclonal antibodies (7E11 and PEQ226.5) in tandem.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein size determination.
- N-terminal amino acid sequencing and cell fractionation.
Main Results:
- PSM' protein was successfully purified from LNCaP cell lysate.
- Purified PSM' exhibited an apparent molecular weight of 95,000 Da, slightly smaller than full-length PSMA (100,000 Da).
- N-terminal sequencing confirmed PSM' begins at residue 60 of PSMA, and it was localized to the cytoplasm of LNCaP cells.
Conclusions:
- The PSM' protein is a distinct, shorter variant of PSMA, differing in its N-terminus.
- PSM' is present in the cytoplasm of LNCaP cells, suggesting potential distinct functions from cell surface PSMA.