scholarly journals Recombinant production of active microbial transglutaminase in E. coli by using self-cleavable zymogen with mutated propeptide

2020 ◽  
Author(s):  
Ryo Sato ◽  
Kosuke Minamihata ◽  
Ryutaro Ariyoshi ◽  
Hiromasa Taniguchi ◽  
Noriho Kamiya
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Recombinant Expression ◽  
Expression System ◽  
Chimeric Protein ◽  
Microbial Transglutaminase ◽  
Cross Linking ◽  
E Coli ◽  
Tev Protease ◽  
Recombinant Expression System

AbstractMicrobial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7±2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.

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 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 Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

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 Reconstitution of the recombinant human γ-tubulin ring complex

Open Biology ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 200325
Author(s):  
Martin Würtz ◽  
Anna Böhler ◽  
Annett Neuner ◽  
Erik Zupa ◽  
Lukas Rohland ◽  
...  
Keyword(s):  
Insect Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Cryo Electron Microscopy ◽  
Egg Extract ◽  
Activation Mechanisms ◽  
Ring Complex ◽  
Core Components ◽  
End Capping ◽  
Recombinant Expression System

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.

scholarly journals Structural Characterization of the 1918 Influenza Virus H1N1 Neuraminidase

2008 ◽  
Vol 82 (21) ◽  
pp. 10493-10501 ◽  
Author(s):  
Xiaojin Xu ◽  
Xueyong Zhu ◽  
Raymond A. Dwek ◽  
James Stevens ◽  
Ian A. Wilson
Keyword(s):  
Influenza Virus ◽  
Recombinant Expression ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Spanish Flu ◽  
1918 Influenza ◽  
Recombinant Expression System ◽  
Influenza Virus Neuraminidase ◽  
Group 1

ABSTRACT Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 “Spanish flu” (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-Å and 1.45-Å resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147 to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.

scholarly journals Cross-linking Phosphatidylinositol-specific Phospholipase C Traps Two Activating Phosphatidylcholine Molecules on the Enzyme

2004 ◽  
Vol 279 (19) ◽  
pp. 20490-20500 ◽  
Author(s):  
Xin Zhang ◽  
Hania Wehbi ◽  
Mary F. Roberts
Keyword(s):  
Phospholipase C ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Cross Linking ◽  
Tryptophan Residue ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Bacterial Model ◽  
Chemical Cross Linking

Bacillus thuringiensisphosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces. Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles. Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC7PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles. PI-PLC cross-linked in the presence of a diC7PC interface exhibited an enhanced specific activity for PI cleavage. The extent of this cross-linking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this (βα)8-barrel protein. The higher activity of the native protein cross-linked in the presence of diC7PC correlated with an increased affinity of the protein for two diC7PC molecules as detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In contrast to wild type protein, W47A and W242A had only a single diC7PC tightly associated when cross-linked in the presence of that activator molecule. These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.

Autophosphorylation of the 16 kDa and 70 kDa antigens (Hsp 16·3 and Hsp 70) of Mycobacterium tuberculosis

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2135-2141 ◽  
Author(s):  
Rachel Preneta ◽  
K. G. Papavinasasundaram ◽  
Alain J. Cozzone ◽  
Bertrand Duclos
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Calcium Ions ◽  
Expression System ◽  
Cross Linking ◽  
Molecular Characteristics ◽  
Two Dimensional ◽  
Hsp 70 ◽  
E Coli ◽  
Environmental Variations ◽  
Vertebrate Eye

Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been previously cloned and are being exploited in the development of improved vaccines and diagnostic reagents. In this study, the molecular characteristics of two of these antigens, the immunodominant proteins Hsp 16·3 and Hsp 70, were analysed in further detail by assessing their capacity to undergo protein phosphorylation, a chemical modification frequently used by organisms to adjust to environmental variations. Hsp 16·3 was overproduced in an Escherichia coli expression system and purified to homogeneity. Upon incubation in the presence of radioactive ATP, it was shown to possess autophosphorylation activity. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at serine residues. In addition, cross-linking experiments demonstrated that it could tightly bind to ATP. Purified Hsp 70 was also shown to autophosphorylate but phosphorylation occurred exclusively at threonine residues. This reaction was found to be strongly stimulated by calcium ions. These data indicate that both structural and functional similarities exist between Hsp 16·3 (Acr) and α-crystallin, a eukaryotic protein which plays an important role in maintaining the transparency of the vertebrate eye, and that the functional properties of Hsp 70 from M. tuberculosis are similar to those of other bacterial members of the Hsp 70 family, particularly the E. coli homologue DnaK.

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