scholarly journals Purification and biochemical characterization of recombinant human placental growth hormone produced in Escherichia coli

1993 ◽  
Vol 295 (3) ◽  
pp. 719-724 ◽  
Author(s):  
A Igout ◽  
J Van Beeumen ◽  
F Frankenne ◽  
M L Scippo ◽  
B Devreese ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Hormone ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Expression System ◽  
Pituitary Hormone ◽  
Native Form ◽  
E Coli ◽  
Placental Growth Hormone

The hGH-V (or hGH-2) gene codes for human placental growth hormone (hPGH). Secretion of hPGH is continuous, in contrast with the pulsed secretion of pituitary growth hormone (hGH) which it progressively replaces in the maternal bloodstream. hGH-V cDNA has previously been cloned and isolated. Analysis of its nucleotide sequence has revealed a 191-residue protein, hPGH, differing from hGH at 13 positions. The calculated pI is more basic than that of the pituitary hormone. Here we have inserted hGH-V cDNA into the pIN-III-ompA3 plasmid in order to produce hPGH in its native form in Escherichia coli D1210. Expression of hGH-V cDNA in E. coli is significantly lower than that of hGH cDNA with the same expression system. The hPGH produced in E. coli was purified in quantities sufficient to allow its biochemical and immunochemical characterization. The molecular mass of the protein was determined by electrospray m.s. The determined mass, 22,320 Da, agrees well with the molecular mass calculated from the translated cDNA sequence, assuming the presence of two disulphide bridges. Having established the technique for producing hPGH with a primary structure identical to the natural, non-glycosylated, 22 kDa isoform, we can now plan the full physicochemical and pharmaceutical characterization of this new hormonal entity.

scholarly journals Characterization of GlnK1 from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1

2002 ◽  
Vol 184 (4) ◽  
pp. 1028-1040 ◽  
Author(s):  
Claudia Ehlers ◽  
Roman Grabbe ◽  
Katharina Veit ◽  
Ruth A. Schmitz
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Ammonium Assimilation ◽  
Regulatory Function ◽  
Native Form ◽  
E Coli ◽  
Methanosarcina Mazei ◽  
Apparent Homogeneity

ABSTRACT Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK1 is encoded in the glnK1 -amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK1 was cloned and overexpressed in E. coli, and GlnK1 was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E. coli GlnK. First, GlnK1 from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.

scholarly journals Molecular Cloning, Purification, and Biochemical Characterization of Hydantoin Racemase from the Legume Symbiont Sinorhizobium meliloti CECT 4114

2004 ◽  
Vol 70 (1) ◽  
pp. 625-630 ◽  
Author(s):  
Sergio Martínez-Rodríguez ◽  
Francisco Javier Las Heras-Vázquez ◽  
Lydia Mingorance-Cazorla ◽  
Josefa María Clemente-Jiménez ◽  
Felipe Rodríguez-Vico
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Molecular Cloning ◽  
Sinorhizobium Meliloti ◽  
Biochemical Characterization ◽  
Competitive Inhibition ◽  
Optimum Temperature ◽  
Aromatic Rings ◽  
Native Form

ABSTRACT Hydantoin racemase from Sinorhizobium meliloti was functionally expressed in Escherichia coli. The native form of the enzyme was a homotetramer with a molecular mass of 100 kDa. The optimum temperature and pH for the enzyme were 40°C and 8.5, respectively. The enzyme showed a slight preference for hydantoins with short rather than long aliphatic side chains or those with aromatic rings. Substrates, which showed no detectable activity toward the enzyme, were found to exhibit competitive inhibition.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals Molecular and Biochemical Characterization of the xlnD-Encoded 3-Hydroxybenzoate 6-Hydroxylase Involved in the Degradation of 2,5-Xylenol via the Gentisate Pathway in Pseudomonas alcaligenes NCIMB 9867

2005 ◽  
Vol 187 (22) ◽  
pp. 7696-7702 ◽  
Author(s):  
Xiaoli Gao ◽  
Chew Ling Tan ◽  
Chew Chieng Yeo ◽  
Chit Laa Poh
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Molecular Mass ◽  
Electron Donor ◽  
Biochemical Characterization ◽  
Aromatic Substrates ◽  
Gentisate Pathway ◽  
A Subunit ◽  
Pseudomonas Alcaligenes

ABSTRACT The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.

scholarly journals Purification and parcial characterization of a hemagglutinating factor (HAF): a possible adhesive factor of the diffuse adherent of Escherichia coli (DAEC)

1996 ◽  
Vol 38 (6) ◽  
pp. 401-406 ◽  
Author(s):  
Yano Tomomasa ◽  
Cleide Ferreira Catani ◽  
Michiko Arita ◽  
Takeshi Honda ◽  
Toshio Miwatani
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Hela Cells ◽  
Immunofluorescence Test ◽  
Native Form ◽  
Bacterial Surface ◽  
E Coli ◽  
Sds Page ◽  
Adhesive Factor

The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains

scholarly journals Overproduction and Biochemical Characterization of the Chryseobacterium meningosepticum BlaB Metallo-β-Lactamase

2002 ◽  
Vol 46 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
Sandrine Vessillier ◽  
Jean-Denis Docquier ◽  
Sandrine Rival ◽  
Jean-Marie Frere ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ion Exchange ◽  
Kinetic Parameters ◽  
Culture Supernatant ◽  
Biochemical Characterization ◽  
Expression System ◽  
T7 Promoter ◽  
Hydrolysis Of

ABSTRACT The BlaB metallo-β-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k cat/Km ratios of >106 M−1 s−1) toward most penam and carbapenem compounds, with the exception of the 6-α-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-β-lactamase inhibitors, including β-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.

scholarly journals Identification and Biochemical Characterization of a Novel Protein Phosphatase 2C-Like Ser/Thr Phosphatase in Escherichia coli

2018 ◽  
Vol 200 (18) ◽  
Author(s):  
Krithika Rajagopalan ◽  
Elizabeth Nagle ◽  
Jonathan Dworkin
Keyword(s):  
Escherichia Coli ◽  
Protein Phosphatase ◽  
Biochemical Characterization ◽  
Regulatory Protein ◽  
Negative Bacterium ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Novel Protein

Regulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria, including the model Gram-negative bacteriumEscherichia coli, demonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thr phosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

scholarly journals Identification and Biochemical Characterization of the Novel α2,3-Sialyltransferase WbwA from Pathogenic Escherichia coli Serotype O104

2015 ◽  
Vol 197 (24) ◽  
pp. 3760-3768 ◽  
Author(s):  
Diana Czuchry ◽  
Paul Desormeaux ◽  
Melissa Stuart ◽  
Donald L. Jarvis ◽  
Khushi L. Matta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Biochemical Characterization ◽  
Enzyme Reaction ◽  
T Antigen ◽  
Glycan Structure ◽  
Content Type ◽  
E Coli ◽  
Pathogenic Escherichia Coli

ABSTRACTThe sialyl-T antigen sialylα2-3Galβ1-3GalNAc is a common O-glycan structure in human glycoproteins and is synthesized by sialyltransferase ST3Gal1. The enterohemorrhagicEscherichia coliserotype O104 has the rare ability to synthesize a sialyl-T antigen mimic. We showed here that thewbwAgene of theE. coliO104 antigen synthesis gene cluster encodes an α2,3-sialyltransferase WbwA that transfers sialic acid from CMP-sialic acid to Galβ1-3GalNAcα-diphosphate-lipid acceptor. Nuclear magnetic resonance (NMR) analysis of purified WbwA enzyme reaction product indicated that the sialyl-T antigen sialylα2-3Galβ1-3GalNAcα-diphosphate-lipid was synthesized. We showed that the conserved His-Pro (HP) motif and Glu/Asp residues of two EDG motifs in WbwA are important for the activity. The characterization studies showed that WbwA fromE. coliO104 is a monofunctional α2,3-sialyltransferase and is distinct from human ST3Gal1 as well as all other known sialyltransferases due to its unique acceptor specificity. This work contributes to knowledge of the biosynthesis of bacterial virulence factors.IMPORTANCEThis is the first characterization of a sialyltransferase involved in the synthesis of an O antigen inE. coli. The enzyme contributes to the mimicry of human sialyl-T antigen and has unique substrate specificity but very little sequence identity to other sialyltransferases. Thus, the bacterial sialyltransferase is related to the human counterpart only by the similarity of biochemical activity.

scholarly journals CULTURAL AND BIOCHEMICAL CHARACTERIZATION OF SHEEP Escherichia coli ISOLATED FROM IN AND AROUND BAU CAMPUS

1970 ◽  
Vol 8 (1) ◽  
pp. 51-55 ◽  
Author(s):  
M Purkayastha ◽  
MSR Khan ◽  
M Alam ◽  
MP Siddique ◽  
F Begum ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Antibiotic Sensitivity ◽  
Resistance Pattern ◽  
E Coli ◽  
Bright Pink ◽  
Healthy Sheep ◽  
Metallic Sheen ◽  
Apparently Healthy

Pathogenic Escherichia coli remain as an important etiological agent of sheep diarrhoea in Bangladesh. The present study was designed for the cultural and biochemical characterization of Sheep E. coli from diarrhoeic and apparently healthy sheep in and around BAU campus for the period from January to October, 2007. Out of 90 faecal samples, 36 from diarrhoeic and 54 from apparently healthy sheep collected from different areas in and around BAU campus, 15 (41.67%) and 21 (38.38%) were found to be positive for E. coli. The cultural characterization of all positive sheep E. coli revealed greenish black colony with metallic sheen in Eosine methylene blue agar, bright pink color smooth transparent colony in MacConkey agar, green color colony in Brilliant green agar, slight pinkish smooth colony in Salmonella-Shigella agar and colorless colony with hemolysis in blood agar. In case of biochemical characterization, all of the isolates showed fermentation of dextrose, sucrose, fructose, maltose and mannitol with the production of acid and gas, negative result to Voges-Proskaure test, positive result to Methyl-red test and differential result to Indole test.The overall prevalence of E. coli was recorded as 80.05% through the cultural and biochemical characterization. The antibiotic sensitivity and resistance pattern showed that the isolates of sheep E. coli were highly sensitive to ciprofloxacine, co-trimoxazol, nalidixic acid and chloramphenicol but to the erythromycin the isolates were highly resistant. DOI = 10.3329/bjvm.v8i1.8350 Bangl. J. Vet. Med. (2010). 8(1): 51-55

scholarly journals Biochemical Characterization of SFC-1, a Class A Carbapenem-Hydrolyzing β-Lactamase

2007 ◽  
Vol 51 (12) ◽  
pp. 4512-4514 ◽  
Author(s):  
Fátima Fonseca ◽  
Ana Cristina Sarmento ◽  
Isabel Henriques ◽  
Bart Samyn ◽  
Jozef van Beeumen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulfate ◽  
Molecular Mass ◽  
Clavulanic Acid ◽  
Gel Electrophoresis ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Class A

ABSTRACT The carbapenem-hydrolyzing β-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam.

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