Overexpression and purification of folded domain of prostate cancer related proteins MSMB and PSA.

Overexpression of domains of a human protein using recombinant DNA technology has been challenging because individual domains intend to accumulate as non-soluble aggregate when expressed separately. Studies on identifying right sequences for a domain to be able to fold independently may help understand the folding pattern and underlying protein-engineering events to isolate the functional domains of a protein. In this report, individual domains of prostate cancer related biomarkers; MSMB and PSA were overexpressed in bacterial system and purified in their folded forms using affinity chromatography. The western blotting experiment using domain specific antibodies further confirmed these proteins. The designed nucleotide sequences domains were truncated using fold index software and folding were predicted by phyre2 and I-TASSER software. Other parameters were optimized for their overexpression and purification using Co-NTA affinity chromatography. Purified domains of each protein showed secondary structures such as α + ß type for PSA, α/ß and ß type for the each domains of PSA and MSMB respectively. This is the first report on producing PSA and MSMB individual domains in functional folded forms. This study may help produce the folded domain of many such proteins to be used for better diagnostic purpose.

Novel inhibitory activity for serine protease inhibitor Kazal type-3 (Spink3) on human recombinant kallikreins.

Kallikrein-related peptidases (KLKs) are trypsin-like and chymotrypsin-like serine proteases which are expressed in several tissues. Their activity is tightly controlled by inhibitors including members of the serine protease Kazal-type (SPINK) family. These enzymes are promising targets for the treatment of skin desquamation, inflammation and cancer. Spink3 or caltrin I is expressed in mouse pancreas and males accessory glands and the resulting mature protein has been associated with different activities such as an inhibitor of trypsin and acrosin activity, calcium transport inhibitor in sperm and inhibitor of cell proliferation during embryogenesis. In this study, we produced a soluble recombinant Spink3 from mouse seminal vesicle (rmSpink3) that inhibited the activity of human KLKs. Using FRET substrates, rmSpink3 exhibited a potent inhibitory activity against human KLK2, KLK3, KLK5 (Ki ranging from 260 to 1500 nM), and to a lesser extent against KLK6, KLK1 and KLK7 (Ki around 3000 nM). As shown by mass spectrometry analysis of rmSpink3 incubated with trypsin, the inhibitor was not truncated by the target enzyme. Based on the in silico analysis of the expression of Spink3/SPINK1 and KLKs it is speculated that some KLKs may be natural targets of Spink3/SPINK1, however experimental confirmation using both proteins from mouse or human origin is needed. This work shows that rmSpink3 is a potent inhibitor of various human KLK members suggesting the potential of this molecule in the diagnosis/prevention of several human diseases.

Purification and preliminary X-ray crystallographic studies of beta-microseminoprotein from human seminal plasma.

beta-Microseminoprotein (beta-MSP) is a small cysteine-rich protein with a molecular mass of 10 kDa. It was first isolated from human seminal plasma and has subsequently been identified from several species. Comparison of the amino-acid sequences of beta-MSP proteins suggests that the protein is a rapidly evolving protein. The function of beta-MSP is poorly understood. Furthermore, no crystal structure has been reported of any beta-MSP; therefore, determination of the crystal structure of beta-MSP is the foremost task in order to understand the function of this protein completely. Here, the purification, crystallization and preliminary X-ray diffraction analysis of beta-MSP from human seminal plasma are described. The protein was purified using anion-exchange and size-exclusion chromatography and the purified protein was crystallized using 0.1 M ammonium sulfate, 0.1 M HEPES buffer pH 7.0 and 20%(w/v) PEG 3350. The crystals belonged to the tetragonal space group P4(3)22 and contained three beta-MSP molecules in the asymmetric unit. X-ray intensity data were collected to 2.4 A resolution.

Beta-microseminoprotein in serum correlates with the levels in seminal plasma of young, healthy males.

Beta-microseminoprotein (MSP) is one of the most abundant proteins secreted by the prostate gland. Because MSP is also synthesized in nonreproductive organs, the establishment of a solid relationship between the levels of MSP in serum and semen is crucial for future studies connecting MSP with aging or diseases of the prostate gland. We developed a specific, competitive, europium-based immunoassay to measure MSP in serum and seminal plasma. We also produced recombinant MSP in insect cells using baculo virus and purified it to homogeneity by a novel approach with ethanol extraction and gel filtration. The median values of MSP in 205 young men were 12 microg/L (2.5-97.5 percentile, 4.9-26 microg/L) in serum and 0.53 g/L (2.5-97.5 percentile, 0.13-2.0 g/L) or 1.8 mg (2.5-97.5 percentile, 0.32-6.6 mg) in seminal plasma. MSP in serum showed significant correlation to MSP in seminal plasma (r = .50, P < .001). Significant correlations were also found in seminal plasma between MSP and prostate-specific antigen (PSA) (r = .65, P < .001) and between MSP and Zn(2+) (r = .54, P < .001). The yield of recombinant MSP in culture medium was 35 mg/L or higher, and recovery following ethanol extraction was 80%-90%. MSP in serum reflects the prostate secretion of MSP, and correlations were also found in seminal plasma between MSP and PSA and Zn(2+). This suggests that MSP in serum can be used as a marker of prostate secretion, despite the contribution from extra prostatic tissues.

Disulphide bond reduction and S-carboxamidomethylation of PSP94 affects its conformation but not the ability to bind immunoglobulin.

Prostate secretory protein of 94 amino acids (PSP94) is a small non-glycosylated, cysteine rich protein with a molecular mass of 10 kDa. It has also been referred to as beta-microseminoprotein (beta-MSP) and proteins homologous to it have been reported in a number of species. Comparison of the amino acid sequence of these proteins suggests that, it is a rapidly evolving protein. However, all the ten cysteine residues are well conserved in these homologues, indicating their possible role in maintaining the structure and function of these proteins. In the present study, PSP94 was purified from human seminal plasma and characterized further and it showed the presence of five disulfide bonds. Reduction of disulphide bonds of PSP94 led to significant changes in the secondary and tertiary structure of PSP94. CD of disulphide bond reduced PSP94 indicates an overall decrease in the beta sheet content from 79.8% to 46.4%. Tertiary structural changes as monitored by fluorescence quenching reveal that reduction of disulphide bonds of PSP94 followed by the modification of the free thiol groups leads to complete exposure of Trp32 and Trp92 and that one or more side chain carboxyl groups move closer to their indole side chains. Antibodies against native and modified PSP94 demonstrated that the changes following reduction of disulphide linkages are within the immunodominant region of the protein. Changes induced in the functional properties of PSP94, if any, by modification were investigated with respect to IgG binding as PSP94 has been reported to be similar to immunoglobulin binding factor purified from seminal plasma. A novel finding from this study is that both native PSP94 as well as modified protein have the ability to bind human IgG, suggesting the involvement of sequential epitopes of PSP94 in IgG binding.

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.

Evidence of a novel biomarker, alphas1-Casein, a milk protein, in benign prostate hyperplasia.

Benign prostate hyperplasia (BPH) is a common disease in elderly men. Although it is a non-malignant disease, it has a significant detrimental impact on the quality of life in patients with late-stage disease. Owing to the lack of specific markers, diagnosis of early-stage BPH has been proven unsuccessful. Recently, using two-dimensional electrophoresis, we identified a group of prostatic secretory proteins that are specifically produced by BPH cells (Xu et al., Electrophoresis 2003; 24: 1311). In this study, we investigated the potential diagnostic value of one of the secretory proteins, alphas1-Casein, in BPH by inmmunohistological staining of normal, BPH and prostate cancer tissues. We found that 90% (20 out of 22) of BPH tissues showed moderate to strong alphas1-Casein protein expression whereas none of the normal tissues (0 out of 10) and less than 10% of the prostate cancer tissues (3 out of 30) showed similar staining intensity. Our results suggest that alphas1-Casein may be a potential biomarker for early identification of BPH patients.

Identification of a specifically expressed modified form of novel PSP-94 protein in the secretion of benign prostatic hyperplasia.

Using two-dimensional gel electrophoresis, we investigated the profile of prostatic secretory proteins in human expressed prostatic secretion (EPS) from benign prostate hyperplasia (BPH) patients and compared the patterns with normal controls. We identified three specifically expressed proteins, including prostate secretory protein 61 (PSP61), in EPS from benign BPH patients but absent in normal controls. In addition, we found that PSP61 was a modified isoform of the well-documented PSP-94, which had a perfect matching (100% homology) to the first 61 amino acids of the PSP-94 protein but with a deleted C-terminus. This shortened PSP61 was not due to alternative splicing of the PSP-94 gene at transcription level. Our results provide first evidence on the possibility of using PSP61 as a specific biological marker for diagnosis of BPH.