Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding

To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.     

Baculovirus expressed C-terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 inhibits ZP3-mediated induction of acrosomal exocytosis

Zona pellucida glycoprotein-3 (ZP3) has been postulated as the primary sperm receptor in various mammalian species including bonnet monkey (Macaca radiata). However, information on the domain responsible for its binding to spermatozoa is inadequate. In the present study, bonnet monkey ZP3 (bmZP3), corresponding to amino acid (aa) residues 223-348 [bmZP3(223-348)] has been cloned and expressed using baculovirus expression system. SDS-PAGE and Western blot analysis of the purified renatured recombinant protein revealed it as a closely spaced doublet of approximately 25 kDa. Lectin-binding studies documented the presence of both O- as well as N-linked glycans. The biotinylated r-bmZP3(223-348) binds to the acrosomal region of the capacitated spermatozoa but fails to bind to the acrosome-reacted spermatozoa as investigated by immunofluorescence studies. In ELISA, nonbiotinylated r-bmZP3(223-348) and baculovirus expressed r-bmZP3, devoid of signal sequence and transmembrane-like domain [r-bmZP3(23-348)] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r-bmZP3(23-348) to the capacitated sperm is also inhibited by nonbiotinylated r-bmZP3(223-348). In contrast to r-bmZP3(23-348), r-bmZP3(223-348) failed to induce acrosomal exocytosis in the capacitated sperm. Interestingly, it competitively inhibits the acrosomal exocytosis induced by r-bmZP3(23-348). These studies, for the first time, identify a domain of ZP3 capable of binding to capacitated spermatozoa and inhibiting ZP3-mediated induction of acrosomal exocytosis furthering our understanding of mammalian fertilization.     

Characterization of the functional domains of boar acrosin involved in nonenzymatic binding to homologous zona pellucida glycoproteins

During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida. To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs). The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody. The recombinant truncated β-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation. Three of the five recombinant forms (truncated β-acrosin, Ser/Ala²²²-truncated β-acrosin, and truncated β-acrosin “heavy chain”) had the ability to bind ZPGPs. The two shorter forms (the amino and carboxy termini of truncated β-acrosin) failed to bind. The catalytic site mutant (Ser/Ala²²²) of truncated β-acrosin does not differ from the recombinant truncated β-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process. Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site. Mol. Reprod. Dev. 49:426–434, 1998. © 1998 Wiley-Liss, Inc.     

Interaction of human spermatozoa with the human zona pellucida and zona-free hamster oocyte following capacitation by exposure to human cervical mucus

The functional capacity for sperm interaction with the human zona pellucida and zona-free hamster oocyte was tested after human spermatozoa were capacitated by passage through a column of human cervical mucus. The results were compared with those obtained when spermatozoa from an aliquot of the same semen sample were capacitated by the standard laboratory methods involving sequential washing by dilution and centrifugation of the semen. Washed-capacitated sperm suspensions were more successful than mucus-capacitated sperm in attaching to the zona-free hamster oocyte and in fusing with its oolemma. However, the ability of mucus-capacitated sperm to penetrate the human zona pellucida was equal to washed capacitated sperm. These experiments demonstrate an approach that may be useful in comparative studies of human sperm capacitation in vivo and in vitro.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

人卵透明带蛋白ZP3a和ZP3b肽段的免疫原性及其抗血清体外抑制人精子-半透明带结合

分析大肠杆菌表达的重组人卵透明带-3（r-huZP3）蛋白两个肽段（r-huZP3a^22-176及r-huZP3b^177-348）的免疫原性,比较两者抗血清体外抑制人精子-半透明带结合的能力。以制备性聚丙烯酰胺凝胶电泳（PAGE）纯化的大肠杆菌表达蛋白为抗原,主动免疫新西兰兔产生抗huZP3a^22-176及huZP3b^177-348抗血清：通过ELISA测定比较两者的抗体应答水平;以蛋白印迹和免疫组化方法测定两者抗血清同重组表达蛋白、大然人卵ZP以及卵巢组织的反应性;通过竞争性半透明带结合试验（hemizona assay,HZA）观察两者抗血清体外抑制人精子-ZP结合能力。结果显示：未与人分子蛋白载体耦联的r-huZP3a^22-176和r-huZP3b^177-348抗原都在免疫兔中产生了较高的抗体滴度,而且它们的抗血清可识别人肠杆菌表达的各自重组ZP3肽以及人卵细胞表面天然ZP,两者抗血清也都能在体外抑制人精子-ZP结合。由此可见,r-huZP3^22-176及r-huZP3b^177-348蛋白具有良好免疫原性,所产生抗血清也都显示出细胞和组织特异性。因此,单一或合并两个huZP3肽段均可作为抗原研制检测不明原因性不孕妇女中是否存在抗自身ZP抗体的诊断试剂盒,另外它们的抗血清也能用于鉴定已知huZP3表位肽段的最小B-细胞表位基序。     

Characterization of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs

Bovine zone pellucida (ZP) glycorproteins from ovarian egg emerged as three bands with molecular mass of 78 kDa, 64 kDa and 21 kDa in SDS-PAGE under reducing conditions. Endo-β-galactosidase (EβG) digestion of the glycoproteins yielded five products with molecular mass of 76 kDa (EβG-76), 68 kDa (EβG-68), 63 kDa(EβG-63), 47 kDa (EβG-47) and 21 kDa (EβG-21) under the same conditions. The N-terminal amino acid sequences of EβG-76 and EβG-21 were identical. This fact together with the results of diagonal SDS-PAGE indicated that EβG-21 (N-terminal region) is linked to EβG-63 (C-terminal region) through disulfide bond to form EβG-76. Immunoblot analysis using anti-pig ZP protein antibodies revealed that bovine EβG-76, EβG-68 and EβG-47 correspond to pig PZP2, PZP3α and PZPEβ glycoproteins, respectively. The EβG-76 and EβG-68 components were shown to be specifically cleaved during fertilization.     

GABA, progesterone and zona pellucida activation of PLA2 and regulation by MEK-ERK1/2 during acrosomal exocytosis in guinea pig spermatozoa

We investigated whether GABA activates phospholipase A2 (PLA2) during acrosomal exocytosis, and if the MEK-ERK1/2 pathway modulates PLA2 activation initiated by GABA, progesterone or zona pellucida (ZP). In guinea pig spermatozoa prelabelled with [14C]arachidonic acid or [14C]choline chloride, GABA stimulated a decrease in phosphatidylcholine (PC), and release of arachidonic acid and lysoPC, during exocytosis. These lipid changes are indicative of PLA2 activation and appear essential for exocytosis since inclusion of aristolochic acid (a PLA2 inhibitor) abrogated them, along with exocytosis. GABA activation of PLA2 seems to be mediated, at least in part, by diacylglycerol (DAG) and protein kinase C since inclusion of the DAG kinase inhibitor R59022 enhanced PLA2 activity and exocytosis stimulated by GABA, whereas exposure to staurosporine decreased both. GABA-, progesterone- and ZP-induced release of arachidonic acid and exocytosis were prevented by U0126 and PD98059 (MEK inhibitors). Taken together, our results suggest that PLA2 plays a fundamental role in agonist-stimulated exocytosis and that MEK-ERK1/2 are involved in PLA2 regulation during this process.     

脑型一氧化氮合成酶的钙调蛋白结合区的表达及活性鉴定

用PCR法克隆出nNOS的CaM结合区基因(nNOS 2455～2988bp),并在大肠杆菌中进行了高效表达。经金属离子螯合亲和层析得到纯度为90％以上的重组蛋白．分子量为22kDa,CaM Oveday assay证实该蛋白具有CaM的结合活性。由于所表达的重组蛋白既具有序列特异性又具有CaM的结合活性．因此。可将它作为筛选nNOS特异性抑制肽的靶蛋白,亦可用于特异性抗体的制备。     

人b防御素3和植物des-pGLu1-brazzein融合蛋白表达菌的诱导条件优化及其活性分析

对人b防御素3和植物甜蛋白des-pGlu1-Brazzein嵌合基因的工程菌株BL-pET-hBD3-Bra的IPTG诱导表达条件进行了研究, 同时对所表达的目的蛋白进行了纯化和活性分析。IPTG浓度、诱导时间和诱导温度对菌株生长和目的蛋白表达的试验结果显示: 所取的IPTG浓度(0.2~1.0 mmol/L)对菌株生长和目的蛋白的表达无显著影响(P>0.05); 菌株的生物量随着诱导时间的延长而增加, 6 h优于4 h(P<0.01), 但是蛋白的表达量无明显增加(P>0.05); 其中温度是重要的影响因素, 在30°C诱导时, 目的蛋白的表达量占总蛋白的35%左右。进一步的研究表明, 菌株在30°C~32°C生长, 在 30°C诱导最优。对目的蛋白的活性分析表明, 所得到的hBD3-Bra融合蛋白有甜味, 其甜度大约是蔗糖的200倍, 但是其杀菌活性很弱, 经凝血酶切割后, des-pGlu1-Brazzein的甜度大大提高, 大约为蔗糖甜度的600倍, 重组hBD3对大肠杆菌和金黄色葡萄球菌有明显的抑菌活性。     

人b防御素3和植物des-pGLu1-brazzein融合蛋白表达菌的诱导条件优化及其活性分析

对人b防御素3和植物甜蛋白des-pGlu1-Brazzein嵌合基因的工程菌株BL-pET-hBD3-Bra的IPTG诱导表达条件进行了研究, 同时对所表达的目的蛋白进行了纯化和活性分析。IPTG浓度、诱导时间和诱导温度对菌株生长和目的蛋白表达的试验结果显示: 所取的IPTG浓度(0.2~1.0 mmol/L)对菌株生长和目的蛋白的表达无显著影响(P>0.05); 菌株的生物量随着诱导时间的延长而增加, 6 h优于4 h(P<0.01), 但是蛋白的表达量无明显增加(P>0.05); 其中温度是重要的影响因素, 在30°C诱导时, 目的蛋白的表达量占总蛋白的35%左右。进一步的研究表明, 菌株在30°C~32°C生长, 在 30°C诱导最优。对目的蛋白的活性分析表明, 所得到的hBD3-Bra融合蛋白有甜味, 其甜度大约是蔗糖的200倍, 但是其杀菌活性很弱, 经凝血酶切割后, des-pGlu1-Brazzein的甜度大大提高, 大约为蔗糖甜度的600倍, 重组hBD3对大肠杆菌和金黄色葡萄球菌有明显的抑菌活性。     

Process optimization for large-scale production of TGF-α-PE40 in recombinant Escherichia coli: effect of medium composition and induction timing on protein expression

The effects of medium composition and induction timing on expression of a chimeric fusion protein TGF-α -PE40 (TP-40) in Escherichia coli strain RR1 were examined using a complex medium at several fermentor scales. Two distinctive phases in E. coli catabolism were identified during fermentation based on preferential utilization between protein hydrolysate and glycerol. Maximum specific and volumetric productivities were achieved by inducing the culture when the cells were switching substrate utilization from protein hydrolysate to glycerol. By increasing the yeast extract concentration in the production medium, initiation of the catabolic switch was delayed until high cell mass was achieved. The final titer of TP-40 at the 15-L fermentation scale was doubled from 400 mg L⁻¹ to 850 mg L⁻¹ by increasing the yeast extract concentration from 1% to 4% (w/v) and delaying the time of induction. This fermentation process was rapidly scaled up in 180-L and 800-L fermentors, achieving TP-40 titers of 740 and 950 mg L⁻¹, respectively. Received 26 August 1996/ Accepted in revised form 10 December 1996     

Conversion of recombinant human ferritin light chain inclusion bodies into uniform nanoparticles in Escherichia coli for facile production

Prokaryotic expression systems are widely used to produce many types of biologics because of their extreme conveniences and unmatchable cost. However, production of recombinant human ferritin light chain (rhFTL) protein is largely restrained because its expression in Escherichia coli tends to form inclusion bodies (IBs). In this study, a prokaryotic expression vector (FTL‐pBV220) harboring the rhFTL gene was constructed using a pBV220 plasmid. The tag‐free rhFTL was highly expressed and almost entirely converted to soluble form, and thus the rhFTL was successfully self‐assembled into uniform nanoparticles in E. coli. To establish a simplified downstream process, a precipitation procedure based on the optimized incubation temperature, pH condition, and ionic strength was developed to remove impurities from the crude lysate supernatant. The rhFTL retained in the clarified supernatant was subsequently purified in a single step using Capto Butyl column resulting in a considerable recovery and high purity. The purified rhFTL was characterized and verified by mass spectrometry and spectral and morphological analyses. The results revealed that rhFTL exhibited highly ordered and fairly compact structures and the spherical structures were preserved.     

Characterization of chicken endoglin, a member of the zona pellucida family of proteins, and its tissue expression

Endoglin is a TGF-β co-receptor expressed in endothelial cells, where it plays a crucial role in angiogenesis, cardiovascular development and vascular remodeling. In humans, mutations in the endoglin gene give rise to Hereditary Hemorrhagic Telangiectasia type 1 (HHT1), an autosomal dominant disorder associated with vascular lesions in skin, mucosa and internal organs. So far, endoglin cDNA has been sequenced in several species from mammals, amphibians and birds. While in mammals the characterization of endoglin protein expression and function is well documented, little is known about the protein homologue in birds. In silico analysis by multiple sequences alignment showed a low homology score of 30-33 between the full length chicken endoglin protein and several mammalian homologues. However, a high homology score (80-85) was observed with the cytoplasmic and transmembrane regions and the overall structure of the zona pellucida (ZP) and orphan domains of the extracellular region appear to be conserved. Transient expression of chicken endoglin allowed the identification of a 180-kDa disulfide linked homodimer similar to the mammalian homologues. To further characterize its tissue expression, the novel specific monoclonal antibody (mAb) 7H5A8 was generated against chicken endoglin transfectant cells. The mAb 7H5A8 specifically recognized chicken endoglin by western blot, immunoprecipitation, immunofluorescence flow cytometry as well as immunofluorescence microscopy assays and displayed a positive staining of the endothelium in veins and arteries from frozen tissue sections of lung and bursa of Fabricius. These results may help to further understand the endoglin expression in vertebrates.