Cloning, expression, purification and functional characterization of recombinant human PSP94.
Human PSP94 (prostate secretory protein of 94 amino acids) is a major protein synthesized by the prostate gland and secreted in large quantities in seminal fluid. Previous studies have suggested a potential biomedical utility of PSP94 in applications such as diagnosis/prognosis and in treatment of human prostate cancer (PCa). This study was designed to produce a recombinant human PSP94 (rPSP94) to evaluate its clinical and functional role in PCa. We cloned PSP94 cDNA and successfully expressed an active recombinant protein in yeast using Pichia pastoris expression system. A simple purification strategy was established that incorporated combination of membrane ultrafiltration (Pellicon tangential-flow system) and anion exchange chromatography using DE52 resin. The method minimized the technical level of expertise for the production of high quality functional protein. The purified rPSP94 (>98% purity) showed a single band with SDS-PAGE analysis and a peak with a molecular mass (M(r)) of 11,495 kDa using MALDI TOF mass spectrometry (MS). The in vitro competitive binding assays indicated high functional similarity of the rPSP94 with that of its native counterpart. Furthermore, in vivo administration of rPSP94 caused a significant growth inhibition of hormone refractory Mat LyLu tumors in Dunning rat model. Taken together, our data provides evidence for high suitability of the purified rPSP94 for evaluation of its potential diagnostic and therapeutic role in PCa and as a valuable analytical reference standard for clinical studies.
Garde
Seema
S
Ambrilia Biopharma Inc. 1000, Chemin du Golf Verdun, Canada.
Fraser
Jennifer E
JE
Nematpoor
Najib
N
Pollex
Rebecca
R
Morin
Catherine
C
Forté
André
A
Rabbani
Shafaat
S
Panchal
Chandra
C
Gupta
Madhulika B
MB
D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006801:Humans; D008297:Male; D010843:Pichia / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000188:drug therapy; D029584:Prostatic Secretory Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000970:Antineoplastic Agents; D029584:Prostatic Secretory Proteins; D011994:Recombinant Proteins; C043076:beta-microseminoprotein