Expression, purification, and characterization of active recombinant prostate-specific antigen in Pichia pastoris (yeast).

BACKGROUND: Prostate-specific antigen (PSA), a member of the kallikrein family of serine proteases, is a chymotrypsin-like glycoprotein produced by the prostate epithelium. Elevated serum PSA (> 4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA over time is indicative of metastatic disease. It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular matrix glycoproteins, as well as cleavage of IGF binding protein-3, a modulator of IGF-1. To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective source of enzymatically active protein. Previous efforts to express recombinant PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide. We describe the expression of active recombinant mature PSA in yeast. METHODS: Stable chromosomal integration of a construct consisting of the yeast alpha-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA. The rPSA was purified from the yeast cell culture supernatant to homogeneity by strong cation-exchange chromatography, and characterized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-glycanase digestion, N-terminal amino acid sequencing, and inactivation by a PSA-specific inhibitor. RESULTS: We report the production of active, mature rPSA in Pichia pastoris. Two forms of rPSA varying slightly in glycosylation were identified. The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 micromol/min mg) as determined using a chromogenic substrate. CONCLUSIONS: Large-scale production of active rPSA will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis. In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing.

In vivo demonstration that human parathyroid hormone 1-38 inhibits the expression of osteoprotegerin in bone with the kinetics of an immediate early gene.

Osteoprotegerin (OPG) is a potent inhibitor of osteoclast formation and function. To elucidate how OPG is regulated in bone, we examined (1) the expression and localization of OPG protein in bone tissue, (2) the effect of human parathyroid hormone 1-38 (hPTH 1-38) on OPG messenger RNA (mRNA) levels in rat femur metaphyseal and diaphyseal bone, and (3) the effect of hPTH(1-38) on expression of OPG mRNA in cultured osteoblast-like cells derived from the metaphysis and diaphysis, and in ROS 17/2.8 osteosarcoma cells. Because PTH has been shown to stimulate osteoblast activity via the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal transduction pathway we also investigated whether PTH action on OPG in vivo is dependent on activation of cAMP/PKA pathway. Immunohistochemistry was used to evaluate OPG protein expression and Northern blot hybridization was used to analyze OPG mRNA expression both in vivo and in vitro. Immunohistochemistry of OPG protein expression in the rat distal femur metaphysis revealed that it was localized predominantly in preosteoblasts, osteoblasts, lining cells, and the osteoid layer, with occasional immunoreactivity in osteocytes and cells of the bone marrow. Subcutaneous (sc) administration of a single injection of hPTH(1-38) at 80 microg/kg induced a rapid and transient decrease in OPG mRNA expression in both metaphyseal and diaphyseal bone. The decrease in OPG message was evident by 1 h and mRNA levels returned to baseline after 3 h. PTH analog PTH(1-31), which stimulates intracellular cAMP accumulation, inhibited OPG expression, whereas PTH analogs (3-34 and 7-34) that do not stimulate cAMP production had no effect on expression. In contrast to PTH, prostaglandin E2 (PGE2) had no effect on OPG mRNA expression in vivo in the metaphyseal bone cells, under conditions in which PGE2 does promote expression of the c-fos gene. The in vivo effects of hPTH(1-38) on OPG mRNA were confirmed in isolated primary osteoblast cultures derived from either metaphyseal or diaphyseal bone as well as in ROS 17/2.8 osteosarcoma cells. We propose that the rapid and transient decrease in OPG expression may initiate a cascade of events resulting in the differentiation of osteoclast progenitor. Such a spatially and temporally programmed effect of PTH might contribute to bone turnover.

Threonine phosphorylations induced by RX-871024 and insulin secretagogues in betaTC6-F7 cells.

Treatment of the pancreatic beta-cell line betaTC6-F7 with an imidazoline compound, RX-871024, KCl, or tolbutamide resulted in increased threonine phosphorylation of a 220-kDa protein (p220) concurrent with enhanced insulin secretion, which can be partially antagonized by diazoxide, an ATP-sensitive potassium (K(ATP)) channel activator. Although phosphorylation of p220 was regulated by cytoplasmic free calcium concentration ([Ca(2+)](i)), membrane depolarization alone was not sufficient to induce phosphorylation. Phosphorylation of p220 also was not directly mediated by protein kinase A, protein kinase C, or insulin exocytosis. Analysis of subcellular fractions indicated that p220 is a hydrophilic protein localized exclusively in the cytosol. Subsequently, p220 was purified to homogeneity, sequenced, and identified as nonmuscle myosin heavy chain-A (MHC-A). Stimulation of threonine phosphorylation of nonmuscle MHC-A by KCl treatment also resulted in increased phosphorylation of a 40-kDa protein, which was coimmunoprecipitated by antibody to MHC-A. Our results suggest that both nonmuscle MHC-A and the 40-kDa protein may play roles in regulating signal transduction, leading to insulin secretion.

Enzymatic characterization of refolded human rhinovirus type 14 2A protease expressed in Escherichia coli.

Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn2+ Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY-p-nitroanilide was found to be cleaved by the 2A protease with a k(cat)/Km ratio of approximately 335 M(-1)s(-1), which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.

Crystal structure of the obese protein leptin-E100.

Mutations in the obese gene (OB) or in the gene encoding the OB receptor(OB-R) result in obesity, infertility and diabetes in a variety of mouse phenotypes. The demonstration that OB protein (also known as leptin) can normalize body weight in ob/ob mice has generated enormous interest. Most human obesity does not appear to result from a mutant form of leptin: rather, serum leptin concentrations are increased and there is an apparent inability to transport it to the central nervous system (CNS). Injection of leptin into the CNS of overfed rodents resistant to peripheral administration was found to induce biological activity. Consequently, for the leptin to act as a weight-lowering hormone in human obesity, it appears that appropriate concentrations must be present in the CNS. This places a premium on understanding the structure of the hormone in order to design more potent and selective agonists. Here we report the crystal structure at 2.4A resolution of a human mutant OB protein (leptin-E100) that has comparable biological activity to wild type but which crystallizes more readily. The structure reveals a four-helix bundle similar to that of the long-chain helical cytokine family.

Purification and characterization of secreted human leptin produced in baculovirus-infected insect cells.

The product of the human ob (obesity) gene, leptin, appears to function in the maintenance of body weight in vivo. When injected into mice, this hormone reduces food consumption and causes weight loss. This work has been done with recombinant leptin (re-leptin) purified and renatured from inclusion bodies in Escherichia coli. We have expressed the human obesity gene encoding the predicted full-length leptin in Spodoptera frugiperda (Sf-9) cells by infection with the recombinant baculovirus system. Protein corresponding to re-leptin was secreted into the culture medium and purified in sufficient quantity for testing biological activity. The secreted re-protein was characterized and found to be unmodified except for correct cleavage of the signal peptide during export from the cells. The resulting molecule is expected to be properly folded and has been purified to a high level of homogeneity. The re-leptin secreted from Sf-9 cells should be an appropriate source of protein for study of the native structure.

Leptin is a four-helix bundle: secondary structure by NMR.

Leptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin (13C/15N -labeled) have been assigned and its secondary structure reveals it to be a four-helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short-helix cytokine family. A three-dimensional model was built verifying the mechanical consistency of the identified elements with a short-helix cytokine core.

Clusterin (Apo J) protects against in vitro amyloid-beta (1-40) neurotoxicity.

Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimer's disease (AD), where it is found associated with amyloid-beta (A beta) plaques. Clusterin also coprecipitates with A beta from CSF, suggesting a physiological interaction with A beta. Given this interaction with A beta, the goal of this study was to determine whether clusterin could modulate A beta neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC-9, a full-length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dose-dependent neuroprotection against A beta (1-40) when tested in primary rat mixed hippocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of A beta (1-40) but not against H2O2 or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid-beta plaque pathology.

Structural and functional properties of natural and chemical variants of apolipoprotein A-I.

Four isoforms of human apolipoprotein A-I (apo A-I): the normal allele product and the corresponding Lys-107 deletion mutant, and apo A-I with sulfoxidized Met-112 and Met-148 residues and the corresponding reduced form, were investigated in their lipid binding properties, structures, and abilities to activate lecithin-cholesterol acyltransferase. All apo A-I isoforms reacted completely with palmitoyloleoylphosphatidylcholine to give reconstituted high density lipoprotein (rHDL) particles with diameters of 96 A. These particles reacted with low density lipoprotein (LDL) and lecithin-cholesterol acyltransferase (LCAT) equally well, except that the Lys-107 deletion mutant was resistant to structural rearrangements in the presence of LDL. The spectral measurements revealed only minor structural differences among the free apo A-I forms or among their rHDL products, but showed a decreased stability of the Lys-107 deletion mutant and the isoform with reduced Met towards denaturation by guanidine hydrochloride. The results demonstrate that these specific alterations of the apo A-I sequence, which change the helix orientation and hydrophobic moment in one or two putative lipid binding regions, are not sufficient to disrupt the overall properties of the apo A-I complexes with lipid nor to impair significantly their ability to activate LCAT.

The number of amphipathic alpha-helical segments of apolipoproteins A-I, E, and A-IV determines the size and functional properties of their reconstituted lipoprotein particles.

The objective of this work was to determine the role of the amphipathic alpha-helical structural units of human apolipoproteins A-I, E, and A-IV in defining the sizes and reactivities with lecithin:cholesterol acyltransferase (LCAT) of their reconstituted lipoprotein particles. We prepared reconstituted high density lipoprotein (rHDL) particles with each of the three apolipoproteins in two weight ratios with lipid: 2.7/0.07/1 and 1.35/0.04/1, palmitoyloleoylphosphatidylcholine/cholesterol/apolipoprotein, by the sodium cholate dialysis procedure; and examined the rHDL product sizes and distributions by nondenaturing gradient gel electrophoresis. The rHDL particles were also incubated with low density lipoprotein (LDL), and with LDL plus LCAT, to observe any structural modifications due to phospholipid transfers to LDL and to cholesterol esterification by LCAT. In addition, we examined the average structural properties of the original rHDL by several fluorescence methods and circular dichroism spectroscopy, and determined their reaction kinetics with LCAT. The results indicate that the diameters of the largest rHDL particles, containing two apolipoproteins per particle, correlate with the maximum number of putative amphipathic alpha-helical segments in their sequences, and that smaller particles of this class may arise from the removal of one or more alpha-helical segments from contact with lipid. Furthermore, the larger particles may be converted into the smaller ones upon loss of phospholipid to LDL, and may form one or two well defined products when reacted with LCAT. In general, the subclasses of particles have distinct spectroscopic properties, consistent with a different apolipoprotein folding in particles containing different proportions of phospholipid to apolipoprotein. Furthermore, the different apolipoprotein structures lead to significant differences in reactivity with LCAT.