scholarly journals Purification and characterization of recombinant tissue kallikrein from Escherichia coli and yeast

1991 ◽  
Vol 276 (1) ◽  
pp. 63-71 ◽  
Author(s):  
J Wang ◽  
J Chao ◽  
L Chao
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Shuttle Vector ◽  
Affinity Column ◽  
Tissue Kallikrein ◽  
E Coli ◽  
Rat Tissue

A full-length rat tissue kallikrein cDNA was constructed by oligonucleotide engineering through an extension of RSK 1105, a partial cDNA clone containing 534 bp of the 3′ end of tissue kallikrein, followed by site-directed mutagenesis to remove the vector sequence from within the chimaeric coding sequence. The cDNA has been cloned both into the plasmid pET3b under the control of the T7 promoter/polymerase system, and into the shuttle vector PYE directed by the alpha-factor promoter. Expression in Escherichia coli was detected by direct radioimmunoassay, and recombinant kallikrein of 36 kDa was identified by Western-blot analysis using both polyclonal and monoclonal antibodies to rat tissue kallikrein, and by autoradiography of 14C-labelled L-amino acid-labelled-protein synthesis in the presence of rifampicin. Expression in yeast was also detected by direct radioimmunoassay, and recombinant kallikrein was identified by Western-blot analysis with a molecular mass of 39 kDa. The recombinant kallikrein from yeast, however, remained mostly inactive. Kallikrein was purified to apparent homogeneity from E. coli by DEAE-Sepharose CL-6B and aprotinin-affinity column chromatography and confirmed by the N-terminal ten-amino-acid sequence, which matched the deduced sequence from the cDNA. Both E. coli and yeast recombinant kallikreins have Tos-Arg-OMe-esterolytic and kininogenase activities similar to those of purified tissue kallikrein. Comparisons were made between recombinant kallikreins and rat tissue kallikrein with respect to size, charge, substrate specificity, susceptibility to inhibitors and immunological properties. Our results open the way for the study of kallikrein structure-function relationships through protein engineering.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

scholarly journals Cloning, expression, purification and characterization of Leishmania tropica PDI-2 protein

2016 ◽  
Vol 11 (1) ◽  
pp. 166-176 ◽  
Author(s):  
Dina Ali ◽  
Abdul-Qader Abbady ◽  
Mahmoud Kweider ◽  
Chadi Soukkarieh
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Disulfide Bonds ◽  
Western Blot Analysis ◽  
Affinity Column ◽  
Assay Result ◽  
Essential Enzyme ◽  
Sequencing And Expression ◽  
Cloned Gene

AbstractIn Leishmania species, protein disulfide isomerase (PDI) is an essential enzyme that catalyzes thiol-disulfide interchange. The present work describes the isolation, cloning, sequencing and expression of the pdI-2 gene. Initially, the gene was amplified from L. tropica genomic DNA by PCR using specific primers before cloning into the expression vector pET-15b. The construct pET/pdI-2 was transformed into BL21(DE3) cells and induced for the protein expression. SDS-PAGE and western blot analysis showed that the expressed protein is about 51 kDa. Cloned gene sequence analysis revealed that the deduced amino acid sequence showed significant homology with those of several parasites PDIs. Finally, recombinant protein was purified with a metal-chelating affinity column. The putative protein was confirmed as a thiol - disulfide oxidoreductase by detecting its activity in an oxidoreductase assay. Assay result of assay suggested that the PDI-2 protein is required for both oxidation and reduction of disulfide bonds in vitro. Antibodies reactive with this 51 kDa protein were detected by Western blot analysis in sera from human infected with L. tropica. This work describes for the first time the enzymatic activity of recombinant L. tropica PDI-2 protein and suggests a role for this protein as an antigen for the detection of leishmaniasis infection.

scholarly journals Evaluation of the immunocontraceptive potential of Escherichia coli-expressed recombinant dog ZP2 and ZP3 in a homologous animal model

Reproduction ◽  
2002 ◽  
pp. 847-857 ◽  
Author(s):  
N Srivastava ◽  
R Santhanam ◽  
P Sheela ◽  
S Mukund ◽  
SS Thakral ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Animal Model ◽  
Fusion Protein ◽  
Western Blot ◽  
Zona Pellucida ◽  
Blot Analysis ◽  
Signal Sequence ◽  
E Coli ◽  
Antibody Titres ◽  
Full Length Transcript

Dog zona pellucida glycoprotein 2 (dZP2), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned and expressed as a polyhistidine fusion protein in Escherichia coli to evaluate the immunocontraceptive efficacy of ZP glycoproteins. The recombinant dZP2 (rec-dZP2) revealed a 70 kDa band corresponding to the full length transcript, as well as several low molecular mass fragments in western blot analysis. In addition to rec-dZP2, E. coli expressed recombinant dog ZP glycoprotein 3 (rec-dZP3), which has also been evaluated for its efficacy to block fertility in a homologous system. Three groups of female dogs (n = 4 per group) were immunized with rec-dZP2 conjugated to diphtheria toxoid (rec-dZP2-DT), rec-dZP3 conjugated to DT (rec-dZP3-DT) and DT alone. Immunization of female dogs with rec-dZP2-DT and rec-dZP3-DT led to generation of antibodies against the respective ZP proteins as well as to DT. Subsequent to mating, the four female dogs immunized with rec-dZP2-DT all conceived, which is indicative of failure of the anti-rec-dZP2 antibodies to block fertility. In the group of dogs immunized with rec-dZP3-DT, three of four animals did not conceive when mated with males of proven fertility. The block in fertility was associated with anti-dZP3 antibody titres. Ovarian histopathology revealed that the block in fertility in the group immunized with rec-dZP3-DT is probably manifested by inhibition in the development of follicles and is due to atretic changes in the zona pellucida. These results, although preliminary, indicate that immunization with dZP3 may be a feasible proposition to control dog populations provided that adequate antibody titres are achieved.

scholarly journals Subcellular Localization of the Intracellular Survival-Enhancing Eis Protein of Mycobacterium tuberculosis

2001 ◽  
Vol 69 (7) ◽  
pp. 4295-4302 ◽  
Author(s):  
John L. Dahl ◽  
Jun Wei ◽  
James W. Moulder ◽  
Suman Laal ◽  
Richard L. Friedman
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intracellular Survival ◽  
Terminal Amino Acid ◽  
Terminal Amino

ABSTRACT Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designatedeis, was found to enhance intracellular survival ofMycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377–384, 2000). Wheneis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eisgene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product ofeis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence presented here suggests it associates with the cell surface and is released into the culture medium. It is produced during human tuberculosis infection and therefore may be an important M. tuberculosis immunogen.

scholarly journals Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in Escherichia coli

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2439
Author(s):  
Song Hee Lee ◽  
Tae-Kyun Oh ◽  
Sung Oh ◽  
Seongdae Kim ◽  
Han Byul Noh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Rapid Detection ◽  
Western Blot Analysis ◽  
Apis Cerana ◽  
Korean Isolate ◽  
Sacbrood Virus ◽  
Positive Reactivity

A Korean isolate of the sacbrood virus infecting Apis cerana (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in Escherichia coli BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 103 copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 104 copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.

scholarly journals Serological cross-reactivity of environmental Escherichia coli strains with Shigella-specific antisera

2018 ◽  
Vol 33 (1-2) ◽  
pp. 29-33 ◽  
Author(s):  
Nafisa Azmuda ◽  
Rabeya Bilkis ◽  
Humaira Akter ◽  
Anowara Begum ◽  
Sirajul Islam Khan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Surface Antigens ◽  
Biochemical Properties ◽  
Cross Reactivity ◽  
Freshwater Ecosystems ◽  
Shigella Species ◽  
Immunogenic Proteins

Many bacteria of clinical and environmental origin show evidence of sharing common surface antigens. The present study aimed for isolation of Escherichia coli strains that were serologically cross-reactive with Shigella species from freshwater ecosystems in Bangladesh by conventional cultural methods. Among twenty eight isolates, two isolates, termed 12(35) and 6(50) showed cross-reactivity with four polyvalent serogroup-specific Shigella antisera using slide agglutination assay. The isolates were identified and charcterized by cultural and biochemical properties and Western blot analysis. The isolates showed typical Escherichia coli cell morphology and cultural and biochemical properties and were identified as Escherichia coli by API 20E tests. Western blot analysis confirmed the isolates as cross-reactive with all the four group-specific Shigella antisera due to presence of immunogenic proteins and LPS. One of the isolates also showed cross-reactivity with multiple type-specific Shigella boydii antisera (monovalent) because of immunogenic proteins. Both the isolates were identified as nonpathogenic due to absence of virulence marker genes of diarrheagenic E. coli variants.This study revealed that a number of bacteria present in the environment could share important Shigella species surface antigens. Naturally occurring nonpathogenic environmental bacteria expressing surface antigens specific for certain types of Shigella could be a good choice for vaccine candidates against shigellosis. Bangladesh J Microbiol, Volume 33, Number 1-2, June-Dec 2016, pp 29-33

scholarly journals Overproduction and Purification of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in Escherichia coli Using Thioredoxin as Fusion

2017 ◽  
Vol 21 (1) ◽  
pp. 1
Author(s):  
Dian Fitria Agustiyanti ◽  
Debbie Sofie Retnoningrum ◽  
Heni Rachmawati ◽  
Asrul Muhamad Fuad
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Soluble Form ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor

Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of  fusion protein was 67.37%  from total protein (229.65  mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence. 

The porcine glucocorticoid receptor: sequencing a 2.1 kb cDNA fragment and raising specific polyclonal antibodies for western blotting, immunoprecipitation and immunohistochemistry

2001 ◽  
Vol 73 (2) ◽  
pp. 267-278 ◽  
Author(s):  
M. Gutscher ◽  
S. Eder ◽  
M. Müller ◽  
R. Claus
Keyword(s):  
Glucocorticoid Receptor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyclonal Antibodies ◽  
Cdna Fragment ◽  
E Coli ◽  
Denatured Protein ◽  
Sds Page ◽  
Receptor Fragment

AbstractGlucocorticoids and their tissue receptors are involved in many metabolic and developmental processes. Until now only two short fragments with a total length of 200 amino acids were known from the glucocorticoid receptor of the pig. Therefore we sequenced the main part (2.1 kb) of the porcine receptor. In addition, we subcloned a cDNA fragment of this sequence coding for 135 aa of the modulatory region in a pET expression vector. The protein fragment was expressed in E. coli as a his-tag fusion protein. In the SDS-PAGE, the crude E. coli extracts showed an enrichment of a 15 kDa protein which corresponds to the estimated molecular weight for the receptor fragment.After lysis and Ni-NTA affinity chromatography under denaturing conditions the protein was further purified either by dialysis (native protein) or by SDS-PAGE (linearized form). Both forms were emulsified together in adjuvant and used for rabbit immunization.The resulting antibodies were characterized by western blot analysis, immunoprecipitation, and additionally by immunohistochemistry. Western blot analysis confirmed the binding of the denatured protein by the antiserum and revealed a high binding affinity. Immunoprecipitation demonstrated that both the occupied and unoccupied forms of the receptor are detected. The specificity of the antiserum for pGCR was additionally demonstrated by immunohistochemistry.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Sign in / Sign up

Export Citation Format

Share Document