Establishment of a mammalian expression system for recombinant [–2]proPSA and a specific antibody against the truncated leader peptide

Abstract

A truncated precursor form of prostate-specific antigen (PSA), [-2]proPSA, is a well-known biomarker for prostate cancer. To develop a biomarker assay, highly purified [-2]proPSA is required as a standard reference and for generation of a specific antibody. In this study, we generated an efficient mammalian expression system for producing a recombinant [-2]proPSA-human kappa constant domain (C ) fusion protein. N-terminal amino acid sequencing using Edman degradation demonstrated that over 95% of the recombinant protein produced is [-2]proPSA, thereby showing for the first time that recombinant [-2]proPSA can be produced as a major fraction. We also generated a recombinant chicken antibody specific to [-2]proPSA but not cross-reactive to recombinant [-7]proPSA-C , [-5]proPSA-C , and PSA purified from human seminal fluid in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Also, the recombinant chicken antibody reacted to recombinant [-2]proPSA protein bound to an anti-PSA antibody coated on the micrometer plate in a sandwich ELISA. All of these results suggest that the N-terminus of the [-2]proPSA-C fusion protein resides on the exterior of the protein, thus allowing exposure to the antibody.

Screening of Pro–Asp Sequences Exposed on Bacteriophage M13 as an Ideal Anchor for Gold Nanocubes

“…Therefore, detection of the PSA is commonly used to diagnose prostate cancer. The gene encoding PSA and anti-PSA scFv was subcloned into the modified pCEP4 vector, followed by the expression and purification of the recombinant PSA protein and anti-PSA scFv, as described previously. , Bacteriophages displaying anti-PSA scFv were amplified using wild-type, VSGSSPDS, and PD helper bacteriophages, as described previously . The bacteriophage that was amplified using the PD helper bacteriophage showed a signal compatible with that obtained using the wild-type helper bacteriophage ­(Figure ).…”