scholarly journals β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry

2019 ◽  
Vol 20 (4) ◽  
pp. 947 ◽  
Author(s):  
Suwapat Kittibunchakul ◽  
Mai-Lan Pham ◽  
Anh-Minh Tran ◽  
Thu-Ha Nguyen
Keyword(s):  
Biochemical Characterization ◽  
Recombinant Expression ◽  
Expression System ◽  
Recombinant Enzyme ◽  
Lactobacillus Helveticus ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Interesting Candidate ◽  
High Concentrations ◽  
Discontinuous Mode

β-Galactosidase encoding genes lacLM from Lactobacillus helveticus DSM 20075 were cloned and successfully overexpressed in Escherichia coli and Lactobacillus plantarum using different expression systems. The highest recombinant β-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system based on the T7 RNA polymerase promoter in E. coli, which is more than 1000-fold or 28-fold higher than the production of native β-galactosidase from L. helveticus DSM 20075 when grown on glucose or lactose, respectively. The overexpression in L. plantarum using lactobacillal food-grade gene expression system resulted in ∼2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in E. coli. The recombinant β-galactosidase from L. helveticus overexpressed in E. coli was purified to apparent homogeneity and subsequently characterized. The Km and vmax values for lactose and o-nitrophenyl-β-d-galactopyranoside (oNPG) were 15.7 ± 1.3 mM, 11.1 ± 0.2 µmol D-glucose released per min per mg protein, and 1.4 ± 0.3 mM, 476 ± 66 µmol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and oNPG, is pH 6.5 and optimum temperatures for these reactions are 60 and 55 °C, respectively. The formation of galacto-oligosaccharides (GOS) in discontinuous mode using both crude recombinant enzyme from L. plantarum and purified recombinant enzyme from E. coli revealed high transgalactosylation activity of β-galactosidases from L. helveticus; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes.

scholarly journals Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3512 ◽  
Author(s):  
Adam Bajinting ◽  
Ho Leung Ng
Keyword(s):  
Tyrosine Kinases ◽  
Recombinant Expression ◽  
Expression System ◽  
Transmembrane Helix ◽  
Size Exclusion ◽  
Complex Signal ◽  
Tyrosine Kinase Domain ◽  
Fibroblast Growth ◽  
Fibroblast Growth Factor Receptors ◽  
E Coli

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs) as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

scholarly journals Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase

2021 ◽  
Author(s):  
Monika Wicka-Grochocka ◽  
Hubert Cieśliński ◽  
Marta Wanarska
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Expression System ◽  
Single Domain ◽  
Pseudomonas Sp ◽  
E Coli ◽  
Komagataella Phaffii ◽  
Pichia Pastoris Yeast ◽  
Psychrotolerant Bacterium

Two recombinant Komagataella phaffii (formerly Pichia pastoris) yeast strains for production of two sequential variants of EstS9 esterase from psychrotolerant bacterium Pseudomonas sp. S9, i.e. αEstS9N (a two-domain enzyme consisting of a catalytic domain and an autotransporter domain) and αEstS9Δ (a single-domain esterase) were constructed. However, only one of recombinant K. phaffii strains, namely Komagataella phaffii X-33/pPICZαestS9Δ, allowed to successfully produce and secrete recombinant αEstS9Δ enzyme outside of the host cell. The purified αEstS9Δ esterase was active towards short-chain p-nitrophenyl esters (C2–C8), with optimal activity for the acetate (C2) ester. The single-domain αEstS9Δ esterase exhibits the highest activity at 60oC and pH 9.5. In addition, the enzyme retains 90% of its activity after 3 hour incubation at 70–90oC. What should be also noted is that αEstS9Δ esterase produced in the K. phaffii expression system has a much higher specific activity (0.069 U/mg of protein) than the recombinant EstS9Δ esterase produced in an E. coli expression system (0.0025 U/mg of protein) (Wicka et al., 2016, Acta Biochim Pol 63: 117–125. https://doi.org/10.18388/abp.2015_1074).

scholarly journals Biochemical Characterization of the Pseudomonas aeruginosa 101/1477 Metallo-β-Lactamase IMP-1 Produced byEscherichia coli

1999 ◽  
Vol 43 (4) ◽  
pp. 902-906 ◽  
Author(s):  
Nezha Laraki ◽  
Nicola Franceschini ◽  
Gian Maria Rossolini ◽  
Pasqualino Santucci ◽  
Cécile Meunier ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biochemical Characterization ◽  
Dipicolinic Acid ◽  
Expression System ◽  
Zinc Content ◽  
E Coli ◽  
Gene Coding ◽  
Structural And Functional Properties ◽  
Spectrum Activity ◽  
Final Yield

ABSTRACT The bla IMP gene coding for the IMP-1 metallo-β-lactamase produced by a Pseudomonas aeruginosaclinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21(DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 metallo-β-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 metallo-β-lactamase was not pH dependent.

scholarly journals Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line

1992 ◽  
Vol 284 (1) ◽  
pp. 53-59 ◽  
Author(s):  
F Fossiez ◽  
G Lemay ◽  
N Labonté ◽  
F Parmentier-Lesage ◽  
G Boileau ◽  
...  
Keyword(s):  
Insect Cell ◽  
Mammalian Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Insect Cell Line ◽  
Cell Types ◽  
Neutral Endopeptidase ◽  
Cell System ◽  
Recombinant Enzyme ◽  
Soluble Form

Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.

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 Metabolomic analysis of riboswitch containing E. coli recombinant expression system

2016 ◽  
Vol 12 (2) ◽  
pp. 350-361 ◽  
Author(s):  
Howbeer Muhamadali ◽  
Yun Xu ◽  
Rosa Morra ◽  
Drupad K. Trivedi ◽  
Nicholas J. W. Rattray ◽  
...  
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Recombinant Expression ◽  
Expression System ◽  
Metabolic Effects ◽  
Metabolomic Analysis ◽  
E Coli ◽  
System P ◽  
Recombinant Expression System ◽  
Green Fluorescent

In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein inEscherichia colicells.

scholarly journals Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

2017 ◽  
Author(s):  
Adam Bajinting ◽  
Ho Leung Ng
Keyword(s):  
Tyrosine Kinases ◽  
Recombinant Expression ◽  
Expression System ◽  
Transmembrane Helix ◽  
Size Exclusion ◽  
Complex Signal ◽  
Tyrosine Kinase Domain ◽  
Fibroblast Growth ◽  
Fibroblast Growth Factor Receptors ◽  
E Coli

ABSTRACTFibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs) as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

scholarly journals Identification, Recombinant Expression, and Characterization of LGH2, a Novel Antimicrobial Peptide of Lactobacillus casei HZ1

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2246 ◽  
Author(s):  
Junfang He ◽  
Xuegang Luo ◽  
Duxin Jin ◽  
Yunyang Wang ◽  
Tongcun Zhang
Keyword(s):  
Amino Acid ◽  
Pathogenic Bacteria ◽  
Recombinant Expression ◽  
Expression System ◽  
Fermented Milk ◽  
Random Coil ◽  
Amino Acid Residues ◽  
Inhibitory Peptide ◽  
Antibiotic Resistant ◽  
E Coli

L. casei HZ1 was identified from Chinese traditional fermented milk, and angiotensin converting enzyme inhibitory peptide was separated from its culture in our previous work. Here, LGH2 was a novel AMP, identified from the genome of L. casei HZ1. Altogether, roughly 52.76% of LGH2 was α -helical, with the remainder in β -strand and random coil in 50% TFE solution tested by CD. The peptide was also an amphipathic and cationic molecule, which was composed of 20 amino acid residues. The similarity of the amino acid sequence between LGH2 and Temporin-RN3 was highest. Then, the peptide successfully expressed in E. coli Rossetta (DE3) pLysS using the SUMO fusion expression system and purified by chromatography technologies. The molecular weight of the peptide was 2448 Da determined by MALDI-TOF MS. Antimicrobial tests showed that the peptide has strong activities against G+ bacteria, special for S. aureus (MIC = 4 μM). The toxicity assay showed that the peptide exhibits a low hemolytic activity against sheep red blood cells. The antimicrobial mechanisms of LGH2 against pathogens were further investigated by dye leakage, CLSM, SEM, and FCM assays. We found that LGH2 can bind to the cell membrane, and destroy its integrity. These significant results indicate that LGH2 has great potential to treat the infections caused by pathogenic bacteria such as S. aureus, and it provides a new template to improve antimicrobial peptides targeting antibiotic-resistant pathogenic bacteria.

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