Recombinant PSP94 (prostate secretory protein of 94 amino acids) demonstrates similar linear epitope structure as natural PSP94 protein.
PSP94 has the potential to be a useful diagnostic marker and therapeutic agent in prostate cancer. Recently, different immunoassay systems for quantitative analysis of PSP94 in clinical samples have been developed, but the epitope structure of PSP94 protein has not been elucidated. In this study, we report an Escherichia coli expression system for recombinant GST-PSP94 fusion protein. GST-PSP94 contains antigenic determinants similar to natural PSP94 protein (determined both by Western blotting experiments and by ELISA) and can be used to study the structure of natural PSP94 antigen. Since GST-PSP94 was expressed in E. coli and purification involved a denaturing process, we propose that the epitope structure of PSP94 is linear and largely dependent on the primary amino acid sequence, rather than conformational structure. This hypothesis was supported by reciprocal competition in ELISA among natural, GST-PSP94 fusion protein, and purified recombinant PSP94 protein. The results demonstrate that the various forms of PSP94 can compete with each other in binding to rabbit PSP94 polyclonal antibody, although the natural PSP94 has a slightly higher affinity. When natural and recombinant PSP94 protein were denatured in vitro with urea and alkali, no effect on the binding to antibody was found. The epitope activity of natural PSP94 was also shown to be resistant to the treatment of detergent and reducing agent. The location of one of the linear epitopes recognized by the PSP94 antibody was determined to be in the N-terminus by using two synthetic peptides representing N- and C-terminal sequences. Competitive ELISA between the N-terminal peptide and PSP94 protein indicate that both natural and GST-PSP94 have similar immunoactive N-termini.
Xuan
J W
JW
Department of Surgery, University of Western Ontario, Canada. jxuan@julian.uwo.ca
Wu
D
D
Guo
Y
Y
Fraser
J E
JE
Chin
J L
JL
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry*; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000378:metabolism; D029584:Prostatic Secretory Proteins; D011506:Proteins / Q000737:chemistry*; D011817:Rabbits; D029589:Seminal Plasma Proteins; D013078:Sperm Immobilizing Agents / Q000737:chemistry*
D000939:Epitopes; D029584:Prostatic Secretory Proteins; D011506:Proteins; D029589:Seminal Plasma Proteins; D013078:Sperm Immobilizing Agents; C043076:beta-microseminoprotein; D005982:Glutathione Transferase