Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH

Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

<div><p>Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma.</p> </div

Two-dimensional gel electrophoresis profile of fraction III.

<p>The 2D gel resolved by isoelectricfocusing in the first dimension (pH range 3–10) followed by SDS-PAGE in the second dimension was stained with silver nitrate. The protein spots (circled) were excised and subjected to MS and MS/MS analysis (Data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058631#pone-0058631-t001" target="_blank">Table 1</a>). Molecular weight markers shown are in kDa.</p

Co-immunoprecipitation of PSP94 and PAP proteins from fraction III.

<p><b>A.</b> PSP94-PAP complex from fraction III (50 µg) co-immunoprecipitated with anti-PAP antibody showing the presence of PSP94 (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with mouse isotype control antibody (lane 1) served as controls. 10 µg of fraction III was loaded in lane 4 as input and the immunoblot was probed with anti-PSP94 antibody. <b>B.</b> PSP94-PAP complex from fraction III (50 µg) was co-immunoprecipitated with anti-PSP94 antibody showing the presence of PAP (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with normal rabbit serum (lane 1) served as controls. 10 µg of fraction III was loaded in lane 3 as input and the immunoblot was probed with anti-PAP antibody. Molecular weight markers shown are in kDa.</p

Identification of proteins in fraction III.

<p><b>A.</b> MALDI-TOF mass spectra of fraction III from preparative RP-HPLC showing a major peak of molecular mass 46753 Da. The peak at 10772 Da is probably of PSP94. <b>B.</b> Immunoblot analysis of fraction III probed with anti-PAP antibody. Molecular weight markers shown are in kDa.</p

Interaction of pure PSP94 and PAP proteins in vitro.

<p>500 ng of PSP94 incubated with or without PAP (500 ng) (lane 1 and 2 respectively) and immunoprecipitated using anti-PAP antibody. Pure PSP94 (20 ng; lane 3) and PAP (500 ng; lane 4) proteins were loaded as input. Lanes 1, 2 and 3 were immunoblotted with anti-PSP94 antibody, while lane 4 was immunoblotted with anti-PAP antibody. The immunoreactive band of PSP94 (∼17 kDa) is detected only in lane 1 and not in lane 2. Molecular weight markers shown are in kDa.</p

Detection of PSP94-PAP complex in human seminal plasma.

<p><b>A.</b> PSP94-PAP complex from seminal plasma (100 µg) was co-immunoprecipitated with anti-PAP antibody (lane 1). Protein G beads incubated with seminal plasma in buffer alone served as the negative control (lane 2). 20 µg of seminal plasma was loaded in lane 3 as input and the immunoblot was probed with anti-PSP94 antibody. The immunoreactive band of PSP94 (∼17 kDa) is detected only in lane 1 and not in lane 2. <b>B.</b> PSP94-PAP complex from seminal plasma (100 µg) was co-immunoprecipitated with anti-PSP94 antibody (lane 1). Protein G beads incubated with seminal plasma in buffer alone served as the negative control (lane 2). 20 µg of seminal plasma was loaded in lane 3 as input and the immunoblot was probed with anti-PAP antibody. The immunoreactive band of PAP (∼47 kDa) is detected only in lane 1 and not in lane 2. Molecular weight markers shown are in kDa.</p

Mass spectrometric analysis of spots excised from the 2D gel.

*<p>Note: Discrepancy in the isoelectric point (pI) or molecular weight (MW) of the proteins detected here with that of the protein entries in NCBI database could be probably due to the post-translational modifications or protein processing.</p

Fractionation of human seminal plasma proteins.

<p><b>A.</b> Preparative RP-HPLC profile of seminal plasma proteins (bound fraction from Phenyl Sepharose chromatography) showing three major peaks (fractions I, II and III). <b>B.</b> Immunoblot of fractions I, II and III probed with anti-PSP94 antibody. <b>C.</b> One dimensional SDS-PAGE profile of fractions I, II and III. Molecular weight markers shown are in kDa.</p