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1.
Bioengineered ; 10(1): 87-97, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30957636

RESUMO

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)20, was engineered either at the N- or C-terminal end of AAT. The (AP)20 tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)20-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)20 along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)20 module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.


Assuntos
Elastase Pancreática/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , alfa 1-Antitripsina/biossíntese , Sequência de Bases , Técnicas de Cultura de Células , Dipeptídeos/genética , Dipeptídeos/metabolismo , Expressão Gênica , Glicosilação , Humanos , Elastase Pancreática/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Células Vegetais/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tabaco/citologia , Tabaco/metabolismo , Transformação Genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/farmacologia
2.
J Nat Prod ; 80(10): 2659-2665, 2017 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-28968119

RESUMO

As part of an ongoing search for new natural products from medicinal plants to treat respiratory disease, six new compounds, a dihydroflavonol (1) and five C-geranylated flavanones (3, 6, 8, 13, and 14), and 13 known compounds were isolated from mature fruits of Paulownia tomentosa. The structures of the new compounds were determined via interpretation of their spectroscopic data (1D and 2D NMR, UV, IR, ECD, and MS). In biological activity assays with human alveolar basal epithelial cells, the expression of TNF-α-induced proinflammatory cytokines (IL-8 and IL-6) was reduced significantly by the EtOAc fraction of a P. tomentosa extract as well as by the new compounds isolated from this fraction. Furthermore, the majority of the isolates (1-19 except 5-7) were found to inhibit human neutrophil elastase (HNE) activity, with IC50 values ranging from 2.4 ± 1.0 to 74.7 ± 8.5 µM. In kinetic enzymatic assays with the HNE substrate MeOSuc-AAPV-pNA, compound 17 exhibited the highest inhibitory activity (Ki = 3.2 µM) via noncompetitive inhibition. These findings suggest that the flavanone constituents of P. tomentosa fruits may be valuable for the development of new drug candidates to treat airway inflammation.


Assuntos
Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Flavanonas/isolamento & purificação , Flavanonas/farmacologia , Frutas/química , Magnoliopsida/química , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Anti-Inflamatórios não Esteroides/química , Flavanonas/química , Humanos , Interleucina-6/análise , Interleucina-8/análise , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Secretadas Inibidoras de Proteinases/química , República da Coreia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/farmacologia
3.
J Enzyme Inhib Med Chem ; 31(sup1): 16-22, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27558014

RESUMO

Campylotropis hirtella is used as a food supplement in the subtropical region of China. In an intensive hunt for human neutrophil elastase inhibitors, we isolated eight flavonoids from C. hirtella three of which (1-3) emerged to be elastase inhibitors. Geranylated flavonoids (1-3) displayed significant inhibitory activity with IC50s between 8.5 and 30.8 µM. The most striking example was geranylated isofavanone 3 that inhibited elastase significantly (IC50 = 30.8 µM) but its parent compound (dalbergioidin) and isoflavone analog (5) were inactive (IC50 > 200 µM). Compounds (1-3) displayed different kinetic mechanisms (noncompetitive, competitive, and mixed type, respectively) that were dependent upon the parent skeleton. The competitive inhibitor, isoflavan-3-ol-4-one 2 manifested an inhibition of isomerization profile for elastase with kinetic parameters K5 = 0.0386 M-1S-1, K6 = 0.0244 µM-1S-1 and Kiapp = 16.3427 µM. The specific identification of metabolites was accomplished by LC-DAD-ESI/MS that was also used to analyze abundance of active components (1-3) within the plant.


Assuntos
Fabaceae/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Elastase de Leucócito/antagonistas & inibidores , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Relação Dose-Resposta a Droga , Flavonoides/síntese química , Flavonoides/química , Humanos , Cinética , Elastase de Leucócito/metabolismo , Estrutura Molecular , Proteínas Secretadas Inibidoras de Proteinases/química , Relação Estrutura-Atividade
4.
J Biol Chem ; 289(41): 28284-98, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-25147180

RESUMO

Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcß4,6S(1 → 4) [FucαX(1 → 3)]GlcAß(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.


Assuntos
Anti-Inflamatórios não Esteroides/química , Sulfatos de Condroitina/química , Doenças do Sistema Imunitário/tratamento farmacológico , Transtornos Leucocíticos/tratamento farmacológico , Peritonite/tratamento farmacológico , Proteínas Secretadas Inibidoras de Proteinases/química , Pepinos-do-Mar/química , Animais , Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Configuração de Carboidratos , Sulfatos de Condroitina/isolamento & purificação , Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/farmacologia , Peróxido de Hidrogênio , Doenças do Sistema Imunitário/metabolismo , Doenças do Sistema Imunitário/patologia , Ferro , Selectina L/química , Selectina L/metabolismo , Transtornos Leucocíticos/metabolismo , Transtornos Leucocíticos/patologia , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Oxirredução , Selectina-P/química , Selectina-P/metabolismo , Peritonite/metabolismo , Peritonite/patologia , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/farmacologia
5.
PLoS One ; 8(12): e82600, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24358212

RESUMO

OBJECTIVE: Epididymal protease inhibitor (Eppin) was located on the surface of spermatozoa and modulates the liquefaction of human semen. Here, we identify the correlative protein partner of Eppin to explore the molecular mechanism of liquefaction of human semen. METHODS: (1) Human seminal vesicle proteins were transferred on the membrane by Western blotting and separated by 2-D electrophoresis and incubated in recombinant Eppin. The correlative protein was identified by Mass Spectrometry (MS) (2). Western blotting was used to determine the relation of rEppin and rFibronectin(Fn); (3) Co-localization in spermatozoa were detected using immunofluorescence; (4) Correalation of Eppin and Fn was proved by co-immunoprecipitation. RESULTS: Fn was identified as the binding partner of recombinant Eppin by MS. Recombinant of Eppin was made and demonstrated that the Eppin fragment binds the fn 607-1265 fragment. The Eppin-Fn complex presents on the sperm tail and particularly in the midpiece region of human ejaculated spermatozoa. Immunoprecipitation indicated that Eppin in the spermatozoa lysates was complexed with Fn. CONCLUSIONS: Our study demonstrates that Eppin and Fn bind to each other in human semen and on human ejaculated spermatozoa. Eppin-Fn complex may involve in semen coagulation, liquefaction and the survival and preparation of spermatozoa for fertility in the female reproductive tract.


Assuntos
Fibronectinas/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Sêmen/metabolismo , Sequência de Aminoácidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Epididimo/metabolismo , Fibronectinas/química , Humanos , Masculino , Dados de Sequência Molecular , Ligação Proteica , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Sêmen/química , Análise do Sêmen , Proteínas Secretadas pela Vesícula Seminal/química , Proteínas Secretadas pela Vesícula Seminal/metabolismo
6.
Prikl Biokhim Mikrobiol ; 49(1): 34-40, 2013 Jan-Feb.
Artigo em Russo | MEDLINE | ID: mdl-23662448

RESUMO

The PKPIJ-B gene encoding a chymotrypsin inhibitor from a subfamily of potato Kunitz-type proteinase inhibitors (PKPI) in potatoes (Solanum tuberosum L. cv. Yubilei Zhukova) was cloned into a pET23a vector and then expressed in Escherichia coli. The recombinant PKPIJ-B protein obtained in the inclusion bodies was denatured, purified by high-performance liquid chromatography (HPLC) on Mono Q under denaturing conditions, and renaturated. The renaturated protein was additionally purified using HPLC on DEAE-ToyoPearl. The PKPIJ-B protein efficiently suppressed chymotrypsin activity, had a weaker effect on trypsin, and inhibited the growth and development of phytopathogenic microorganisms affecting potato plants.


Assuntos
Expressão Gênica , Proteínas de Plantas , Proteínas Secretadas Inibidoras de Proteinases , Solanum tuberosum/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Solanum tuberosum/genética
7.
Protein Expr Purif ; 82(1): 144-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22212898

RESUMO

Human serine proteinase inhibitor Kazal-type 6 (SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6 has been shown to selectively inhibit Kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor. Here, we report the successful generation of tagless SPINK6 using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and size-exclusion chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. Pichia pastoris-derived recombinant human SPINK6 (rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase 14 (KLK14) (K(i)=0.16 nM) than previously reported Escherichia coli-derived rhSPINK6 (K(i)=0.5 nM). This protein also exhibited moderate inhibition of bovine trypsin (K(i)=33 nM), while previous E. coli-derived rhSPINK6 did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.


Assuntos
Pichia/genética , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia em Gel , Expressão Gênica , Humanos , Calicreínas/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Inibidores de Serinopeptidase do Tipo Kazal , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transformação Genética , Tripsina/metabolismo
8.
J Biol Chem ; 285(42): 32174-81, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20667819

RESUMO

Kallikrein-related peptidases (KLKs) play a central role in skin desquamation. They are tightly controlled by specific inhibitors, including the lymphoepithelial Kazal-type inhibitor (LEKTI) encoded by SPINK5 and LEKTI-2 encoded by SPINK9. Herein, we identify SPINK6 as a selective inhibitor of KLKs in the skin. Unlike LEKTI but similar to LEKTI-2, SPINK6 possesses only one typical Kazal domain. Its mRNA was detected to be expressed at low levels in several tissues and was induced during keratinocyte differentiation. Natural SPINK6 was purified from human plantar stratum corneum extracts. Immunohistochemical analyses revealed SPINK6 expression in the stratum granulosum of human skin at various anatomical localizations and in the skin appendages, including sebaceous glands and sweat glands. SPINK6 expression was decreased in lesions of atopic dermatitis. Using KLK5, KLK7, KLK8, KLK14, thrombin, trypsin, plasmin, matriptase, prostasin, mast cell chymase, cathepsin G, neutrophil elastase, and chymotrypsin, inhibition with recombinant SPINK6 was detected only for KLK5, KLK7, and KLK14, with apparent K(i) values of 1.33, 1070, and 0.5 nm, respectively. SPINK6 inhibited desquamation of human plantar callus in an ex vivo model. Our findings suggest that SPINK6 plays a role in modulating the activity of KLKs in human skin. A selective inhibition of KLKs by SPINK6 might have therapeutic potential when KLK activity is elevated.


Assuntos
Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Calicreínas/antagonistas & inibidores , Inibidores de Proteases , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Serina Endopeptidases/metabolismo , Pele/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Humanos , Isoenzimas/genética , Calicreínas/metabolismo , Dados de Sequência Molecular , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Alinhamento de Sequência , Inibidores de Serinopeptidase do Tipo Kazal , Pele/anatomia & histologia , Dermatopatias/metabolismo , Dermatopatias/patologia , Distribuição Tecidual
10.
J Microbiol Biotechnol ; 19(10): 1139-41, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19884771

RESUMO

Clitocybin D, a novel human neutrophil elastase inhibitor, was isolated from the culture broth of Clitocybe aurantiaca. This compound was purified by solvent extraction, silica gel column chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. The compound was determined to be 4-(4,6-dihydroxy-3-methoxy-3H-isoindol-1-yl)-benzoic acid on the basis of 1D and 2D NMRs and MS spectroscopic analysis. Analysis of the human neutrophil elastase (HNE) inhibitory activity of the isolated compound revealed that it showed significant HNE inhibitory activity with an IC(50) value of 17.8 micronM.


Assuntos
Agaricales/química , Meios de Cultura/química , Isoindóis/isolamento & purificação , Elastase de Leucócito/antagonistas & inibidores , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Agaricales/metabolismo , Meios de Cultura/metabolismo , Humanos , Isoindóis/química , Elastase de Leucócito/metabolismo , Ligação Proteica , Proteínas Secretadas Inibidoras de Proteinases/química
12.
Reproduction ; 136(5): 559-71, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18715980

RESUMO

We report a secreted serine protease inhibitor Kazal-type-like (SPINKL) protein. The SPINKL protein was purified from mouse seminal vesicle secretions through a series of steps, including ion-exchange chromatography on a diethylaminoethyl-Sephacel column, gel filtration on a Sephadex G-75 column, and ion-exchange HPLC on a Q strong anion exchange column. Further analysis identified several SPINKL proteins with various N-linked carbohydrates. The SPINKL protein has six conserved cysteine residues that are nearly identical to those of members of the SPINK protein family. It was noted that the SPINKL protein showed no inhibitory activities against common serine proteases such as trypsin, chymotrypsin, subtilisin, or elastase. Spinkl mRNA and SPINKL proteins were found to be primarily expressed in seminal vesicles. Immunohistochemistry revealed that the SPINKL protein occurred in the luminal fluid and mucosal epithelium of the seminal vesicles and was regulated by testosterone. The SPINKL protein was able to bind onto sperm and enhance sperm motility. Also, it was able to suppress BSA-stimulated sperm capacitation and block sperm-oocyte interactions in vitro, suggesting that SPINKL may be a decapacitation factor.


Assuntos
Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Glândulas Seminais/química , Inibidores de Serino Proteinase/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Western Blotting/métodos , Líquidos Corporais/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Feminino , Fertilidade/efeitos dos fármacos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , RNA Mensageiro/análise , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
Am J Physiol Cell Physiol ; 295(2): C423-31, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18550704

RESUMO

Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and carcinoma cells in which hepatocyte growth factor activator inhibitor 1 (HAI-1), a membrane-bound, Kunitz-type serine protease inhibitor, is also expressed. HAI-1 plays dual roles in the regulation of matriptase, as a conventional protease inhibitor and as a factor required for zymogen activation of matriptase. As a consequence, activation of matriptase is immediately followed by HAI-1-mediated inhibition, with the activated matriptase being sequestered into HAI-1 complexes. Matriptase is also expressed by peripheral blood leukocytes, such as monocytes and macrophages; however, in contrast to epithelial cells, monocytes and macrophages were reported not to express HAI-1, suggesting that these leukocytes possess alternate, HAI-1-independent mechanisms regulating the zymogen activation and protease inhibition of matriptase. In the present study, we characterized matriptase complexes of 110 kDa in human milk, which contained no HAI-1 and resisted dissociation in boiling SDS in the absence of reducing agents. These complexes were further purified and dissociated into 80-kDa and 45-kDa fragments by treatment with reducing agents. Proteomic and immunological methods identified the 45-kDa fragment as the noncatalytic domains of matriptase and the 80-kDa fragment as the matriptase serine protease domain covalently linked to one of three different secreted serpin inhibitors: antithrombin III, alpha1-antitrypsin, and alpha2-antiplasmin. Identification of matriptase-serpin inhibitor complexes provides evidence for the first time that the proteolytic activity of matriptase, from those cells that express no or low levels of HAI-1, may be controlled by secreted serpins.


Assuntos
Leite Humano/metabolismo , Serina Endopeptidases/metabolismo , Serpinas/metabolismo , Sequência de Aminoácidos , Antitrombina III/análise , Antitrombina III/isolamento & purificação , Antitrombina III/metabolismo , Western Blotting , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Leite Humano/química , Dados de Sequência Molecular , Proteínas Secretadas Inibidoras de Proteinases/análise , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Serina Endopeptidases/análise , Serina Endopeptidases/isolamento & purificação , Serpinas/análise , Serpinas/isolamento & purificação , alfa 1-Antitripsina/análise , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/metabolismo , alfa 2-Antiplasmina/análise , alfa 2-Antiplasmina/isolamento & purificação , alfa 2-Antiplasmina/metabolismo
14.
Chin Med J (Engl) ; 121(7): 620-4, 2008 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-18466682

RESUMO

BACKGROUND: Eppin (epididymis protease inhibitor) appears to play an important role in primate fertility. However, the function of Eppin and its antibody in men and its relationship with men's infertility are poorly studied. To reveal the significance and possibility of detection of anti-Eppin antibody in clinical infertilty cases, we developed an Escherichia coli expression system for the expression of biologically active human Eppin. METHODS: The human Eppin gene was cloned into PET-28a( )+ vector after induction with 0.5 mmol/L isopropy-beta-D-thiogalactoside (IPTG) at 26 degrees C for 4 hours, and the expressed fusion protein His6-Eppin was purified by Ni2+ affinity chromatography. Afterwards, six female 8-week-old Balb/c mice were immunized with purified His6-Eppin for three weeks. Their sera were collected and polyclonal antibodies against His6-Eppin were purified, all of which were further verified by Western-blot and immunofluorescence analysis. RESULTS: About 18.33 mg His6-Eppin was obtained from 1-L flask culture. The produced polyclonal antibodies against His6-Eppin recognized the Eppin protein both in human epididymis and in HEK293T cells by over-expression of the recombinant human Eppin. CONCLUSION: The purified His6-Eppin protein has biological activity, which might be a candidate for clinical diagnosis of infertility and development of male immuno-contraceptive agents.


Assuntos
Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Escherichia coli/genética , Feminino , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Secretadas Inibidoras de Proteinases/imunologia , Proteínas Recombinantes de Fusão/imunologia
15.
Protein Expr Purif ; 57(1): 45-56, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17936012

RESUMO

The multidomain proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor) consists of 15 potential serine proteinase inhibitory domains. In various diseases such as the severe skin disorder Netherton syndrome as well as atopy, defects in the gene encoding LEKTI have been identified that generate premature termination codons of translation, suggesting a specific role of the COOH-terminal part of LEKTI in healthy individuals. We overexpressed and purified a sequence comprising the 15th domain of LEKTI for further characterisation. Here, we present a high yield expression system for recombinant production and efficient purification of LEKTI domain 15 as a highly soluble protein with a uniform disulfide pattern that is identical to that of other known Kazal-type inhibitors. Also, the expected P1P1' site was confirmed. LEKTI domain 15 is a well-structured protein as verified by circular dichroism (CD) spectroscopy and a tight-binding and stable inhibitor of the serine proteinase trypsin. These findings confirm the designation of domain 15 as a proteinase inhibitor of the Kazal family.


Assuntos
Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , DNA Complementar , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Inibidor de Serinopeptidase do Tipo Kazal 5 , Inibidores de Serino Proteinase/genética , Inibidores de Serino Proteinase/isolamento & purificação , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta , Transformação Bacteriana , Tripsina/metabolismo
17.
Biol Reprod ; 77(3): 476-84, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17567961

RESUMO

Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells. In this study, we have isolated native eppin from ejaculate supernatants (seminal plasma) and washed ejaculate spermatozoa using column chromatography and two-dimensional SDS-PAGE, and identified by mass spectrometry and Western blots an eppin protein complex (EPC) containing lactotransferrin (LTF; also known as lactoferrin), clusterin (CLU), and semenogelin (SEMG1). To confirm the association of eppin with LTF, CLU, and SEMG1, antibodies to CLU and LTF were used to immunoprecipitate CLU and LTF from human sperm lysates. In both cases identical results were obtained, namely, the immunoprecipitate of the EPC. Additionally, we localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, implying that the EPC is present on spermatozoa from the time they leave the seminiferous tubule. On ejaculate spermatozoa eppin, LTF, and CLU colocalize on the tail. The identification of the EPC components suggests that LTF, CLU, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC, implicating the complex as a central player in a network of protein-protein interactions on the human sperm surface. The EPC may provide a surface network with microbicidal properties that protects spermatozoa as well as regulates the sperm's transition to a motile, capacitated sperm.


Assuntos
Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Sêmen/química , Espermatozoides/química , Western Blotting , Cromatografia em Gel , Cromatografia por Troca Iônica , Clusterina/química , Clusterina/isolamento & purificação , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica , Lactoferrina/química , Lactoferrina/isolamento & purificação , Masculino , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas pela Vesícula Seminal/química , Proteínas Secretadas pela Vesícula Seminal/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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