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1.
Fish Shellfish Immunol ; 55: 717-28, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27368542

ABSTRACT

Apolipoprotein A-I (ApoA-I) is the most abundant and multifunctional high-density lipoprotein (HDL) having a major role in lipid transport and potent antimicrobial activity against a wide range of microbes. In this study, a complete CDS of 771 bp of Labeo rohita (rohu) ApoA-I (LrApoA-I) encoding a protein of 256 amino acids was amplified, cloned and sequenced. Tissue specific transcription analysis of LrApoA-I revealed its expression in a wide range of tissues, with a very high level of expression in liver and spleen. Ontogenic study of LrApoA-I expression showed presence of transcripts in milt and 3 h post-fertilization onwards in the larvae. The expression kinetics of LrApoA-I was studied upon infection with three different types of pathogens to elucidate its functional significance. Its expression was found to be up-regulated in the anterior kidney of L. rohita post-infection with Aeromonas hydrophila. Similarly following poly I:C (poly inosinic:cytidylic) stimulation, the transcript levels increased in both the anterior kidney and liver tissues. Significant up-regulation of LrApoA-I expression was observed in skin, mucous, liver and anterior kidney of the fish challenged with the ectoparasite Argulus siamensis. Immunomodulatory effect of recombinant LrApoA-I (rApoA-I) produced in Escherichia coli was demonstrated against A. hydrophila challenge in vivo. L. rohita administered with rApoA-I at a dose of 100 µg exhibited significantly higher protection (∼55%) upon challenge with A. hydrophila 12 h post-administration of the protein, in comparison to that observed in control group, along with higher level of expression of immune-related genes. The heightened expression of ApoA-I observed post-infection reflected its involvement in immune responses against a wide range of infections including bacterial, viral as well as parasitic pathogens. Our results also suggest the possibility of using rApoA-I as an immunostimulant, particularly rendering protection against A. hydrophila.


Subject(s)
Apolipoprotein A-I/genetics , Cyprinidae , Ectoparasitic Infestations/veterinary , Fish Diseases/genetics , Fish Proteins/genetics , Gram-Negative Bacterial Infections/veterinary , Aeromonas hydrophila/physiology , Amino Acid Sequence , Animals , Apolipoprotein A-I/chemistry , Apolipoprotein A-I/metabolism , Arguloida/physiology , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/metabolism , Ectoparasitic Infestations/genetics , Ectoparasitic Infestations/immunology , Ectoparasitic Infestations/parasitology , Fish Diseases/immunology , Fish Diseases/microbiology , Fish Diseases/parasitology , Fish Proteins/chemistry , Fish Proteins/metabolism , Gram-Negative Bacterial Infections/genetics , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/microbiology , Phylogeny , Poly I-C/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment/veterinary
2.
Fish Shellfish Immunol ; 47(2): 913-22, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26518505

ABSTRACT

Cytokines are important components of both adaptive and innate immunity, and are required to initiate and regulate immune responses following infection. The ontogeny and tissue specific distribution of four pro-inflammatory cytokines, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), IL-8 and IL-1ß in rohu (Labeo rohita), and their responses by leucocytes from anterior-kidney/head-kidney (HKLs), spleen (SPLs) and peripheral blood (PBLs) following stimulation with concanavalin A (ConA), ConA with phorbol 12-myristate 13-acetate (ConA/PMA) and formalin-killed Aeromonas hydrophila cells (FAH) were studied. In ontogeny study, mRNA levels of IL-6 and IL-1ß were evident in unfertilized egg stages of L. rohita whereas IL-8 and TNF-α transcripts were found from 1 to 3 h post-fertilization (hpf) onwards till day 15 post-fertilization, respectively. Basal level of all four cytokines was observed in all twelve tissues (eye, brain, heart, gill, anterior kidney, posterior kidney, spleen, liver, skin, muscle, hindgut and foregut) of L. rohita juveniles. Expression levels of IL-6 and IL-8 were found to be the highest in liver and heart tissues, respectively, while TNF-α transcripts were high in anterior kidney and liver tissues. Transcripts of IL-1ß showed high expression in muscle, heart and spleen. Upon in vitro stimulation of leucocytes, there was variable up-regulation of all the four cytokines following different treatments throughout the experimental time period. Induction of cytokines was more pronounced in PBLs stimulated with FAH compared to other stimuli. However, an up-regulated IL-8 expression was evident in all the leucocytes following stimulation with FAH thus indicating IL-8 could be used as an indicator or indirect marker to monitor vaccine status or health status of L. rohita during bacterial infection.


Subject(s)
Cyprinidae , Cytokines/genetics , Fish Diseases/immunology , Fish Proteins/genetics , Gene Expression Regulation, Developmental , Gram-Negative Bacterial Infections/veterinary , Leukocytes/immunology , Aeromonas hydrophila/physiology , Animals , Cytokines/metabolism , Fish Diseases/microbiology , Fish Proteins/metabolism , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/microbiology , Leukocytes/metabolism , Organ Specificity
3.
Fish Shellfish Immunol ; 39(2): 512-23, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24937805

ABSTRACT

Despite the importance and success of developing a candidate vaccine against Aeromonas hydrophila infection in fish, little is known about the molecular mechanisms of the vaccine-induced immunoprotection in Indian major carp, Labeo rohita, primarily due to lack of information on most of the immune related genes of the species. In this study, a novel candidate antigen recombinant outer membrane protein R (rOmpR) of A. hydrophila was evaluated as a vaccine candidate along with a modified adjuvant formulation. Protective efficacy of the rOmpR immunization was assessed in terms of survival against A. hydrophila challenge as well as modulation of immune response in vaccinated fish after 1, 3, 6, 12, 24, 72 h and 10 days post-injection (using immune gene expression analysis) and 10, 28, 56 and 140 days post-injection (serum immune parameter analysis). The generated immune response was compared with a formalin-killed A. hydrophila antigen preparation using mineral oil only and modified adjuvant alone. We report a variable up-regulation of the immune-related genes viz., lysozyme G, complement factor 4, immunoglobulin M, ß2-microglobulin, major histocompatibility complex I and II, and interleukin-1ß in anterior kidney and spleen tissues at early time points post-immunization in all the groups, when compared to the control fish. The vaccinated fish also showed an increase in serum natural hemolysin titer, lysozyme and myeloperoxidase activities, and antibody titer irrespective of vaccine formulations as compared to control fish on days 10, 28 and 56. However, the increase in the serum parameters was more pronounced on day 140 in rOmpR-modified adjuvant injected group, indicating the modulatory role of this new vaccine formulation. Upon challenge with live A. hydrophila on days 56 and 140 post-immunization, significantly reduced percent mortality was noted in the group immunized with modified adjuvant based rOmpR vaccine formulation. Taken together, our results suggest that rOmpR along with modified adjuvant could potentially be used as a vaccine formulation to handle A. hydrophila infection on a long-term basis.


Subject(s)
Aeromonas hydrophila/immunology , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/immunology , Carps , Fish Diseases/immunology , Gram-Negative Bacterial Infections/veterinary , Analysis of Variance , Animals , DNA Primers/genetics , DNA, Complementary/genetics , Gram-Negative Bacterial Infections/immunology , Immunity, Innate/immunology , Muramidase/blood , Peroxidase/blood
7.
Protein Expr Purif ; 18(2): 182-92, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10686149

ABSTRACT

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.


Subject(s)
Human Growth Hormone/metabolism , Inclusion Bodies/metabolism , Animals , Bioreactors , Buffers , Cell Division , Chromatography, Gel , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Escherichia coli/metabolism , Fermentation , Human Growth Hormone/chemistry , Human Growth Hormone/genetics , Human Growth Hormone/isolation & purification , Humans , Hydrogen-Ion Concentration , Inclusion Bodies/chemistry , Inclusion Bodies/genetics , Protein Conformation , Protein Renaturation , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Solubility
8.
FEBS Lett ; 463(3): 336-40, 1999 Dec 17.
Article in English | MEDLINE | ID: mdl-10606749

ABSTRACT

The effects of addition of a few amino acids to the amino- and carboxy-terminal regions of the mature portion of the heat-labile enterotoxin chain B (LTB) of Escherichia coli on protein export, secretion and assembly were investigated. In E. coli, LTB (secretory protein) with or without the extension at the N- or C-terminus accumulated in the periplasmic fraction. For Vibrio cholerae, LTB with the extension at the C-terminus was exported to the periplasm followed by secretion to the extracellular milieu. However, LTB with the N-terminus extension was exported to the periplasm only. Our findings suggest that in the case of V. cholerae, the N-terminus of the mature LTB plays an important role in its secretion to the extracellular milieu.


Subject(s)
Bacterial Toxins/chemistry , Enterotoxins/chemistry , Escherichia coli Proteins , Vibrio cholerae/metabolism , Amino Acids/chemistry , Bacterial Toxins/biosynthesis , Bacterial Toxins/genetics , Base Sequence , Blotting, Western , Enterotoxins/biosynthesis , Enterotoxins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Molecular Sequence Data , Periplasm/metabolism , Receptors, Cell Surface/metabolism , Vibrio cholerae/genetics
9.
Protein Expr Purif ; 17(2): 215-23, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10545269

ABSTRACT

For expression of ovine growth hormone (OGH) in inclusion bodies without an affinity histidine tag at either end of the protein, three clones, differing only in the second codon following the ATG start site, were constructed. Their expression was studied by SDS-PAGE followed by immunoblotting. Clone Ala.OGH (clone 1), beginning with Met.Ala.Phe.Pro ellipsis, did not show any expression. Clone Phe.OGH (clone 3), beginning with Met.Phe.Pro ellipsis, gave very high levels of OGH expression following IPTG induction. However, in clone Gly.OGH (clone 2), in which the Ala codon was replaced with a Gly codon at the second position after the start site, a lower level of expression was obtained. Northern hybridization analysis showed that upon IPTG induction, OGH mRNA was transcribed from all three clones. These results therefore, imply that lack of expression in clone 1 and a lower level of expression in clone 2 are not due to a failure of transcription; however, they may be due to inefficient initiation of translation. The secondary structure analysis of mRNA predicts inaccessibility of different elements of the RBS in the case of Ala.OGH (clone 1). The present study highly underscores the importance of mRNA secondary structure at the start site in regulation of expression of a cloned gene in Escherichia coli, a prokaryotic expression system.


Subject(s)
Codon, Initiator/genetics , Codon/pharmacology , Escherichia coli/genetics , Gene Expression Regulation/drug effects , Growth Hormone/genetics , Sheep/genetics , Animals , Codon/genetics , Gene Expression Regulation/genetics , Genetic Vectors/chemistry , Growth Hormone/biosynthesis , Growth Hormone/drug effects , Mutagenesis, Site-Directed , Protein Biosynthesis , RNA, Messenger/analysis , RNA, Messenger/drug effects , Sheep/physiology , Transcription, Genetic/drug effects , Transformation, Bacterial
10.
DNA Seq ; 10(2): 97-9, 1999.
Article in English | MEDLINE | ID: mdl-10376210

ABSTRACT

The proximal promoter for bubaline lactoferin-encoding gene has been isolated, cloned and sequenced. A 468 bp fragment of the 5' flanking region of the lactoferrin gene was PCR amplified and cloned into pUC18 vector. Sequence analysis of the amplified fragment revealed the presence of one TATA box, one TATA like element, two GC boxes and one motif resembling cAMP response element (CRE) in this region. Bubaline lactoferrin promoter shares 93%, 53%, 52% and 48% homology with cattle, pig, mouse and human lactoferrin 5' flanking region, respectively.


Subject(s)
Buffaloes/genetics , Lactoferrin/genetics , Promoter Regions, Genetic , Animals , Base Sequence , Cattle , Cloning, Molecular , Humans , Mice , Molecular Sequence Data , Swine
11.
DNA Seq ; 10(2): 101-3, 1999.
Article in English | MEDLINE | ID: mdl-10376211

ABSTRACT

The cDNA for Bubalus bubalis growth hormone (GH) has been cloned and sequence determined through RT-PCR approach. The nucleotide sequence of bubaline GH cDNA was in a single reading frame coding for a protein of 191 residues comprising a putative signal sequence of 27 amino acids. Homology comparison of the sequence with other mammalian GH cDNAs showed a very high degree of evolutionary conservation. Bubaline GH sequence shared a homology of 99.5%, 99.5%, 98.6%, 87.6% and 61.9% with that of ovine, caprine, bovine, porcine and human, respectively at amino acid level.


Subject(s)
Buffaloes/genetics , Growth Hormone/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA, Complementary , Humans , Molecular Sequence Data , Sequence Analysis, DNA
12.
DNA Seq ; 10(2): 105-8, 1999.
Article in English | MEDLINE | ID: mdl-10376212

ABSTRACT

The cDNA for bubaline beta-lactoglobulin (beta lg) has been cloned through RT-PCR approach and sequenced. Sequence data showed a single open reading frame coding for a protein of 180 amino acids with a signal sequence of 18 amino acid residues. Comparison with other ruminant beta lg sequences revealed a high homology indicating the protein to be conserved through evolution. The degree of homology, at amino acid level, is 96.1%, 95.6%, 93.9% and 63.7% with goat, sheep, cow and pig, respectively.


Subject(s)
Buffaloes/genetics , Lactoglobulins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA, Complementary , Molecular Sequence Data , Sequence Analysis, DNA
13.
Protein Expr Purif ; 14(2): 155-9, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9790876

ABSTRACT

A single-step method to dissociate histones as well as nonhistone chromosomal proteins from chicken erythrocyte chromatin and their separation into histones H1, H5, core histones, and high mobility group proteins by column chromatography on phosphocellulose is presented. NaCl at 2.0 M is effective in dissociating both histones and nonhistone proteins. The core histones elute as a complex. The pH is a critical factor in separating H5 from the core histones.


Subject(s)
Cellulose/analogs & derivatives , High Mobility Group Proteins/isolation & purification , Histones/isolation & purification , Animals , Cellulose/metabolism , Chickens , Chromatography/methods , Chromosomal Proteins, Non-Histone/isolation & purification , Electrophoresis, Polyacrylamide Gel , Erythrocytes/chemistry
14.
Acta Otolaryngol ; 117(4): 553-8, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9288211

ABSTRACT

Differing levels of the Ca(2+)-ATPase enzymes that reside on the plasma membrane (PM) and on the endoplasmic reticulum (ER) were identified in individual rat cochlear tissues by the use of a semi-quantitative enzyme-linked immunosorbent assay (ELISA). Unlike other studies, a specific antibody to PM Ca(2+)-ATPase was used to detect significantly greater levels (about 2x) of PM Ca(2+)-ATPase in the stria vascularis (SV) than that in the spiral ligament (SL) and organ of Corti (OC) tissues. Similarly, levels of ER Ca(2+)-ATPase were also significantly higher in the SV than in the SL and OC tissues. The presence of ER Ca(2+)-ATPase in the tissues of the SV has not been demonstrated previously. Given the importance of Ca2+ homeostasis in the inner ear, the statistically significantly higher densities of both PM and ER Ca(2+)-ATPase measured in the SV relative to the SL and OC regions would indicate tissue-specific responses to fluctuations in systemic and local Ca2+ concentrations.


Subject(s)
Calcium-Transporting ATPases/metabolism , Cochlear Duct/enzymology , Animals , Antibodies, Monoclonal , Extracellular Space/enzymology , Guinea Pigs , Hair Cells, Auditory, Outer/enzymology , Male , Organ of Corti/enzymology , Rats , Regression Analysis , Stria Vascularis/enzymology
15.
Eur Arch Otorhinolaryngol ; 254(4): 165-8, 1997.
Article in English | MEDLINE | ID: mdl-9151013

ABSTRACT

Free calcium concentration (CCa2+) profiles were evaluated in perilymph, endolymph, marginal cells, spiral ligament and blood serum of adrenalectomized (ADX) rats. Free CCa2+ was significantly greater in perilymph and significantly reduced in the serum of the ADX animals as compared to sham-operated animals. In addition, higher levels of free CCa2+ were found in the spiral ligament in ADX animals. Free CCa2+ did not appear to be affected by ADX in marginal cells and endolymph. These data suggest that marked reductions in endogenous levels of corticosteroids may have a systematic effect on free CCa2+ that is detectable in blood serum as well as cochlear fluids and tissues.


Subject(s)
Adrenal Cortex Hormones/physiology , Calcium Channels/physiology , Calcium/physiology , Cochlear Duct/physiology , Adrenalectomy , Animals , Capillary Permeability/physiology , Endolymph/physiology , Membrane Potentials/physiology , Microelectrodes , Perilymph/physiology , Rats
16.
Protein Expr Purif ; 11(2): 201-8, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9367817

ABSTRACT

The gene for ovine growth hormone (oGH) was expressed without signal sequences in Escherichia coli. A recombinant plasmid expression vector has been constructed which directs the synthesis of a fusion protein containing a stretch of six histidine residues (His6) at the amino-terminus under the control of a T5 promoter. Upon induction with isopropyl-beta-D-thiogalactopyranoside, the recombinant protein was synthesized and accumulated in the cytoplasm in the form of inclusion bodies, at levels of approximately 18% of the total cellular protein. The recombinant ovine growth hormone containing His tag was recovered and purified to >95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity Ni2+.NTA resin that has selectivity for proteins with neighboring histidine residues. Characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and amino terminal analysis demonstrated the authenticity of the fusion protein. The purified RoGH after refolding was found to be functionally active in terms of its receptor binding and antigenicity as analyzed by radio receptor assay and radio immuno assay. Yields of the purified expressed protein were found to be 32 microg/ml at a shake-flask level. Thus, results indicate that a combination of E. coli expression and affinity purification by Ni2+.NTA chromatography promises to be a rapid method to produce oGH for use in structure-function studies.


Subject(s)
Growth Hormone/isolation & purification , Sheep , Amino Acid Sequence , Animals , Chromatography, Affinity , Cloning, Molecular , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Genetic Vectors , Growth Hormone/metabolism , Histidine , Inclusion Bodies , Isopropyl Thiogalactoside/pharmacology , Molecular Sequence Data , Peptides/genetics , Peptides/isolation & purification , Radioimmunoassay , Receptors, Somatotropin/metabolism , Recombinant Fusion Proteins/isolation & purification , Sequence Analysis
17.
Hear Res ; 102(1-2): 63-9, 1996 Dec 01.
Article in English | MEDLINE | ID: mdl-8951451

ABSTRACT

The spontaneously hypertensive rat model has been used to show that hypertension is an important pathophysiological risk factor in age-related hearing loss. In the present study, compound action potential (CAP), electrochemical potential (ECP), and potassium concentration (CK+) measurements were taken from the cochlea of genetically predisposed, spontaneously hypertensive rats (SHR) and from normotensive Wistar-Kyoto (WKY) rats. In the SHR model, as the duration of hypertension increased with the animal's age (from 3 to 8 months), CAP thresholds increased, ECP increased in marginal cells only, and CK+ increased in both endolymph and marginal cells. Collectively, the data suggest that ionic alternations of cellular potentials are involved in hearing changes in the hypertensive state. Ultimately, such data may assist in understanding hearing loss in individuals who are diagnosed with hypertension.


Subject(s)
Aging/pathology , Cochlea/pathology , Cochlear Microphonic Potentials/physiology , Hearing Loss, Sensorineural/etiology , Hypertension/physiopathology , Potassium/metabolism , Action Potentials/physiology , Analysis of Variance , Animals , Auditory Threshold/physiology , Blood Pressure/physiology , Body Weight/physiology , Calibration , Cochlea/metabolism , Disease Models, Animal , Electrophysiology , Homeostasis , Rats , Rats, Inbred SHR , Rats, Inbred WKY
18.
Biochem Biophys Res Commun ; 226(3): 735-40, 1996 Sep 24.
Article in English | MEDLINE | ID: mdl-8831683

ABSTRACT

A gene coding for epsilon-toxin was isolated from a field isolate of Clostridium perfringens type D by PCR amplification and was cloned under the control of T5 promoter fused with six-histidine tag at the amino terminal end. Escherichia coli cells harbouring this construct expressed high levels of the recombinant protein in the form of inclusion bodies. The protein was purified using single step affinity chromatography on a Ni(2+)-nitrilotriacetic acid (NTA) agarose column. Upon immunization of rabbit with the purified recombinant protein, high antibody titre was detected. The antibodies raised against the recombinant protein were able to recognize the recombinant as well as the native toxin. Anti epsilon-toxin monoclonal antibody was able to detect the recombinant protein in a Western blot. N-terminal sequence of the recombinant protein matched with the known sequence of the toxin. At the shake flask level, up to 20 mg of pure epsilon-prototoxin was produced per litre of culture.


Subject(s)
Bacterial Toxins/biosynthesis , Clostridium perfringens/genetics , Clostridium perfringens/metabolism , Animals , Antibodies, Bacterial/biosynthesis , Antibody Formation , Bacterial Toxins/immunology , Bacterial Toxins/isolation & purification , Bacterial Vaccines , Blotting, Western , Chromatography, Affinity , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Gene Expression , Genes, Bacterial , Histidine , Promoter Regions, Genetic , Rabbits , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Restriction Mapping , Sequence Tagged Sites , Vaccines, Synthetic
19.
Gene ; 173(2): 271-4, 1996 Sep 16.
Article in English | MEDLINE | ID: mdl-8964513

ABSTRACT

The PCR-amplified beta-subunit of the human chorionic gonadotropin structural gene (betahCG) was cloned under the control of the tac promoter and the heat-labile enterotoxin chain B (LTB) signal sequence (LTBss). BetahCG was successfully produced, processed and exported to the periplasmic space in Escherichia coli. Expression of betahCG was confirmed by immunoblot analysis using an anti-betahCG polyclonal antibody. The processing of the protein was very efficient, as only the processed band could be detected at all time points during the course of induction. Expression was evident soon after the addition of the lactose analogue, IPTG. These results demonstrate that E. coli cells can synthesize, process and export betahCG using the LTBss.


Subject(s)
Bacterial Toxins/metabolism , Chorionic Gonadotropin, beta Subunit, Human/genetics , Enterotoxins/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Protein Sorting Signals/metabolism , Bacterial Toxins/genetics , Biological Transport , Chorionic Gonadotropin, beta Subunit, Human/metabolism , Enterotoxins/genetics , Escherichia coli/metabolism , Gene Expression/drug effects , Genes , Humans , Isopropyl Thiogalactoside/pharmacology , Plasmids , Protein Processing, Post-Translational , Protein Sorting Signals/genetics
20.
FEBS Lett ; 387(2-3): 167-70, 1996 Jun 03.
Article in English | MEDLINE | ID: mdl-8674542

ABSTRACT

The use of Vibrio cholerae as a secretory expression system for the expression of a mammalian protein, namely human growth hormone, under the control of the heat labile enterotoxin chain B signal sequence is reported. The protein is efficiently expressed and processed. The mature protein is exported to the periplasm after which it is secreted to the extracellular milieu. The expressed and secreted hGH actively binds to its receptor as established by its receptor binding activity. The biological activity of the protein is demonstrated in vitro in a Nb2 proliferation assay.


Subject(s)
Growth Hormone/genetics , Vibrio cholerae/genetics , Animals , Cloning, Molecular , Gene Expression , Growth Hormone/metabolism , Humans , Kinetics , Protein Processing, Post-Translational , Radioimmunoassay , Rats , Receptors, Somatotropin/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
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