120 IDENTIFICATION OF THE RELATIVELY PREDOMINANT BUFFALO INTERFERON tau ISOFORM AND ITS EXPRESSION IN ESCHERICHIA COLI

2014 ◽  
Vol 26 (1) ◽  
pp. 174 ◽  
Author(s):  
S. Saugandhika ◽  
H. N. Malik ◽  
D. K. Singhal ◽  
R. Singhal ◽  
A. Dubey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Western Blot ◽  
Signal Sequence ◽  
Rna Extraction ◽  
Coli Strain ◽  
Soluble Form ◽  
Interferon Tau ◽  
Amino Acid Homology ◽  
Ruminant Species

The maternal pregnancy recognition factor interferon tau (IFN-τ) is expressed in multiple isoforms in all pecoran ruminant species. Interferon-τ, as the first pregnancy signaling molecule, performs a significant role in implantation as well as establishment of pregnancy. Due to low reproductive efficiency of buffalo compared with bovine and IFN-τ being the key molecule of reproductive physiology in ruminants, the objective of our study was framed to identify the various IFN-τ transcripts in buffalo embryonic trophoblast cells, to know their relative abundance to identify the relatively predominant isoform, and lastly to clone and express it in a heterologous host. Following total cellular RNA extraction from primary trophectodermal cells, RT-PCR was performed using gene-specific primers designed against known bovine IFN-τ sequence. Cloning of the amplified product and screening of the recombinant colonies gave 13 distinct cDNA variants that encoded for 8 distinct buffalo IFN-τ isoforms. These buffalo IFN-τ isoforms have a greater nucleotide and amino acid homology with caprine IFN-τ (98–100% and 96–100%) than ovine (94–97% and 90–95%) and bovine (89.6–90.6% and 82–86%), respectively. The novel buffalo IFN-τ isoforms showed pronounced nucleotide and amino acid sequence identity with one another (99.1–99.8% and 98–99%) but only moderate identity with previously identified buffalo IFN-τ (90–92% and 82–86%). All the 13 transcript sequences were accepted in GenBank. Out of 8 isoforms, buffalo IFN-τ1 has been found to be the relatively predominant, which was subcloned into expression vector pET 22b without signal sequence from pJET cloning vector and expressed in competent BL21 (DE3) Escherichia coli strain. Expression of the recombinant protein in soluble form was induced by isopropyl β-D-1-thiogalactopyranoside (0.1 mM) at 30°C for 6 h. The recombinant BuIFN-τ obtained was confirmed by Western blot using anti-HIS antibody. A new 20-kDa protein was detected coinciding the molecular weight of IFN-τ reported earlier in literature. In conclusion, the current study revealed that there are 8 different isoforms of IFN-τ that are expressed in trophectodermal out-growths during early pregnancy of buffalo. Predominantly found isoform IFN-τ 1 was expressed in pET 22b vector, and recombinant soluble protein was confirmed by Western blot.

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 Fermentative Production of l-Alanyl-l-Glutamine by a Metabolically Engineered Escherichia coli Strain Expressing l-Amino Acid α-Ligase

2007 ◽  
Vol 73 (20) ◽  
pp. 6378-6385 ◽  
Author(s):  
Kazuhiko Tabata ◽  
Shin-ichi Hashimoto
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Batch Cultivation ◽  
Coli Strain ◽  
Dependent Manner ◽  
Manufacturing Method ◽  
Fermentative Production ◽  
Final Strain ◽  
Efficient Manufacturing

ABSTRACT In spite of its clinical and nutritional importance, l-alanyl-l-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing l-amino acid α-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of the dpp and pep genes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologous l-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lal and ald expressed under the control of the uspA promoter) or pPE177 (lal and ald expressed under the control of the rpoH promoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containing d-amino acids were formed.

scholarly journals Optimizations of expression and purification of recombinant HIV-1 CRF01_AE p24 protein in Escherichia coli for development of immunodiagnostic assay

2015 ◽  
Vol 24 (1) ◽  
pp. 14-8
Author(s):  
Ade P.R. Simaremare ◽  
Budiman Bela ◽  
Andi Yasmon ◽  
Fera Ibrahim
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
Coli Strain ◽  
Expression And Purification ◽  
Purification System ◽  
P24 Protein ◽  
Native Condition ◽  
Different Strains ◽  
Hiv 1 ◽  
Induction Temperature

Background: Conventional method for confirmation of HIV infection is Western blot. However, it has limitations because of contamination by human cellular antigen and genetic diversity among the HIV-1 subtypes that show indeterminate result and inaccuracy for the diagnosis of different strains. Most of Western blot developed are based on HIV-1 B subtype. In Indonesia HIV-1 CRF01_AE subtype is dominantly circulated. Therefore, we optimized the expression, purification of the recombinant HIV-1 CRF01_AE p24 protein for development of immunodiagnostic assay.Methods: Optimization of protein expression in Escherichia coli strain BL21CP was performed including induction time, isopropyl-1-thio-d-galactopyranoside (IPTG) and immidazole consentrations, and induction temperature. Purification of the recombinant p24 protein was used by using Ni-NTA (Qiagen) purification system in native condition. Results: Expression and purification of HIV-1 CRF01_AE p24 protein have been performed. Confirmation of the recombinant protein by Western blot showed the expression and purification of recombinant p24 protein has been optimized well and reactive with sera of patients with HIV-1 CRF01_AE subtype positive.Conclusion: The recombinant HIV-1 CRF01_AE p24 protein has been expressed and purified successfully, and it is potential to be used as antigen for immunodiagnostic assay.

scholarly journals Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1592
Author(s):  
Monika Cserjan-Puschmann ◽  
Nico Lingg ◽  
Petra Engele ◽  
Christina Kröß ◽  
Julian Loibl ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Recombinant Expression ◽  
Biochemical Properties ◽  
Soluble Form ◽  
Single Chain ◽  
Terminal Amino Acid ◽  
Expression In Escherichia Coli ◽  
Terminal Amino ◽  
Caspase 2

Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at −20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.

A novel class C beta-lactamase (FOX-2) in Escherichia coli conferring resistance to cephamycins.

1997 ◽  
Vol 41 (9) ◽  
pp. 2041-2046 ◽  
Author(s):  
A Bauernfeind ◽  
S Wagner ◽  
R Jungwirth ◽  
I Schneider ◽  
D Meyer
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Amino Acid ◽  
Tract Infection ◽  
Coli Strain ◽  
Beta Lactamase ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Pcr Product

An Escherichia coli strain resistant to a broad spectrum of beta-lactams, including cephamycins, was isolated from a patient suffering from urinary tract infection. A resistance plasmid (pMVP-7) was transferred from the clinical isolate to an Escherichia coli recipient. Both strains produce a cefoxitin-hydrolyzing beta-lactamase focusing at pI 6.7. The phenotype was similar to that of a Klebsiella pneumoniae strain producing cephamycinase FOX-1, so primers were selected from the FOX-1 sequence to amplify the bla gene of the transconjugant. The PCR product obtained was sequenced. The percentage of identity of the deduced amino acid sequence with sequences of other AmpC-type beta-lactamases was 96.9% with FOX-1, 74.9% with CMY-1, and 67.7% with MOX-1. This new plasmid-mediated enzyme is most closely related to FOX-1 (11 amino acid exchanges). We therefore propose the designation FOX-2.

scholarly journals Isolation and Expression in Escherichia coli ofcslA and cslB, Genes Coding for the Chondroitin Sulfate-Degrading Enzymes Chondroitinase AC and Chondroitinase B, Respectively, from Flavobacterium heparinum

2000 ◽  
Vol 66 (1) ◽  
pp. 29-35 ◽  
Author(s):  
Ana Lydia Tkalec ◽  
Dominique Fink ◽  
Françoise Blain ◽  
Guiyi Zhang-Sun ◽  
Maryse Laliberte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Chondroitin Sulfate ◽  
Dna Sequences ◽  
Dermatan Sulfate ◽  
Signal Sequence ◽  
Open Reading Frames ◽  
Amino Acid Residues ◽  
Chondroitin Ac Lyase ◽  
Flavobacterium Heparinum

ABSTRACT In medium supplemented with chondroitin sulfate,Flavobacterium heparinum synthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC 4.2.2.5 ) and chondroitinase B (chondroitin B lyase; no EC number), into its periplasmic space. Chondroitinase AC preferentially depolymerizes chondroitin sulfates A and C, whereas chondroitinase B degrades only dermatan sulfate (chondroitin sulfate B). The genes coding for both enzymes were isolated from F. heparinum and designated cslA (chondroitinase AC) and cslB(chondroitinase B). They were found to be separated by 5.5 kb on the chromosome of F. heparinum, transcribed in the same orientation, but not linked to any of the heparinase genes. In addition, the synthesis of both enzymes appeared to be coregulated. The cslA and cslB DNA sequences revealed open reading frames of 2,103 and 1,521 bp coding for peptides of 700 and 506 amino acid residues, respectively. Chondroitinase AC has a signal sequence of 22 residues, while chondroitinase B is composed of 25 residues. The mature forms of chondroitinases AC and B are comprised of 678 and 481 amino acid residues and have calculated molecular masses of 77,169 and 53,563 Da, respectively. TruncatedcslA and cslB genes have been used to produce active, mature chondroitinases in the cytoplasm of Escherichia coli. Partially purified recombinant chondroitinases AC and B exhibit specific activities similar to those of chondroitinases AC and B from F. heparinum.

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 Cloning and Expression of Islandisin, a New Thermostable Subtilisin from Fervidobacterium islandicum, in Escherichia coli

2005 ◽  
Vol 71 (7) ◽  
pp. 3951-3958 ◽  
Author(s):  
Carolin Gödde ◽  
Kerstin Sahm ◽  
Stan J. J. Brouns ◽  
Leon D. Kluskens ◽  
John van der Oost ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Serine Protease ◽  
Signal Sequence ◽  
Thermophilic Bacterium ◽  
Cloning And Expression ◽  
Inverse Pcr ◽  
Heat Denaturation ◽  
Catalytic Residues

ABSTRACT A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80°C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80°C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80°C with a half-life of 4 h at 90°C and 1.5 h at 100°C.

Plant pyruvate-dependent gamma-aminobutyrate transaminase: identification of an Arabidopsis cDNA and its expression in Escherichia coli

2002 ◽  
Vol 80 (9) ◽  
pp. 933-941 ◽  
Author(s):  
Owen R Van Cauwenberghe ◽  
Amina Makhmoudova ◽  
Michael D McLean ◽  
Shawn M Clark ◽  
Barry J Shelp
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Expressed Sequence Tag ◽  
Signal Sequence ◽  
Functional Expression ◽  
Phosphate Binding ◽  
Leaf Extracts ◽  
Reading Frame ◽  
Nicotiana Tabacum L

Both pyruvate- and 2-oxoglutarate-dependent gamma-aminobutyrate transaminase (GABA-T) activities are present in crude tobacco (Nicotiana tabacum L.) leaf extracts. In this study, GABA:pyruvate-T activity was partially purified using mitochondrial isolation and protein solubilization in 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and a combination of chromatographic and electrophoretic procedures. A partial amino acid sequence of the putative 55-kDa GABA-T subunit enabled identification of a predicted Arabidopsis thaliana (L.) Heynh. GABA:pyruvate-T expressed sequence tag and subsequent amplification of a 1515 bp open reading frame encoding a 504-amino acid polypeptide. Computer analysis using web-based tools revealed the presence of a putative mitochondrial signal sequence and a pyridoxal-5-phosphate binding domain in the polypeptide. Functional expression of the GABA-T cDNA in Escherichia coli revealed that the recombinant protein uses pyruvate but not 2-oxoglutarate. The Arabidopsis GABA:pyruvate-T cDNA could form the basis for identification of multiple GABA-T isoforms and generation of GABA-T mutants for determining the fate of GABA nitrogen and elucidating the physiological function of GABA in plants.Key words: amino acceptor, gamma-aminobutyrate, gamma-aminobutyrate transaminase, protein purification, heterologous expression, recombinant protein.

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