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Establishment of a mammalian expression system for recombinant [–2]proPSA and a specific antibody against the truncated leader peptide
Author(s) -
Hwang Dobeen,
Yoon Aerin,
Kim Soohyun,
Kim Hyori,
Chung Junho
Publication year - 2016
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1481
Subject(s) - recombinant dna , antibody , edman degradation , fusion protein , microbiology and biotechnology , peptide , chemistry , antigen , biology , peptide sequence , biochemistry , immunology , gene
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 κ ) 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 κ , [–5]proPSA‐ C κ , 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 κ fusion protein resides on the exterior of the protein, thus allowing exposure to the antibody.

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