In Vitro Folding of β-1,4Galactosyltransferase and Polypeptide-α-N-Acetylgalactosaminyltransferase from the Inclusion Bodies

2013 ◽  
pp. 321-333 ◽  
Author(s):  
Boopathy Ramakrishnan ◽  
Pradman K. Qasba
Keyword(s):  
Inclusion Bodies

scholarly journals Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates

2021 ◽  
Vol 7 (5) ◽  
pp. 325
Author(s):  
Laura Isabel de de Eugenio ◽  
Rosa Peces-Pérez ◽  
Dolores Linde ◽  
Alicia Prieto ◽  
Jorge Barriuso ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Veratryl Alcohol ◽  
Dye Decolorization ◽  
Irpex Lacteus ◽  
Type D ◽  
Lignin Peroxidases

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

scholarly journals Positive in vitro wound healing effects of functional inclusion bodies of a lipoxygenase from the Mexican axolotl

2018 ◽  
Vol 17 (1) ◽  
Author(s):  
Anne Stamm ◽  
Sarah Strauß ◽  
Peter Vogt ◽  
Thomas Scheper ◽  
Iliyana Pepelanova
Keyword(s):  
Wound Healing ◽  
Inclusion Bodies ◽  
Mexican Axolotl ◽  
Functional Inclusion

scholarly journals In vivo and in vitro folding of a recombinant metalloenzyme, phosphomannose isomerase

1996 ◽  
Vol 318 (2) ◽  
pp. 437-442 ◽  
Author(s):  
Amanda E. I. PROUDFOOT ◽  
Laurence GOFFIN ◽  
Mark A PAYTON ◽  
Timothy N. C. WELLS ◽  
Alain R BERNARD
Keyword(s):  
Inclusion Bodies ◽  
Guanidine Hydrochloride ◽  
Expression Level ◽  
Phosphomannose Isomerase ◽  
Independent Manner ◽  
Body Protein ◽  
Fourfold Increase ◽  
E Coli

Phosphomannose isomerase (PMI) catalyses the interconversion of mannose 6-phosphate and fructose 6-phosphate in prokaryotic and eukaryotic cells. The enzyme is a metalloenzyme which contains 1 mol of zinc per mol of enzyme. Heterologous expression of the cDNA coding for the Candida albicans enzyme in the prokaryotic host Escherichia coli results in an expression level of up to 30% of total E. coli protein. Ten percent of recombinant PMI is expressed in the soluble fraction and 90% in inclusion bodies. Inclusion of a high level of zinc in the fermentation medium resulted in a fourfold increase in soluble protein. Co-expression of the bacterial chaperones, GroES and GroEL, resulted in a proportional twofold increase in soluble PMI while causing an overall decrease in the PMI expression level. Folding denatured PMI in vitro required reductant and zinc ions. The yield of renatured protein was increased by folding in the presence of GroEL and DnaK in an ATP-independent manner. The refolding yield of denatured soluble enzyme from a guanidine solution was threefold higher than that of folding monomerized inclusion body protein solubilized in guanidine hydrochloride. This suggests that a proportion of recombinant protein expressed in E. coli inclusion bodies may be irreversibly denatured.

THE PRODUCTION BY BACILLUS THURINGIENSIS BERLINER OF A HEAT-STABLE SUBSTANCE TOXIC FOR INSECTS

1959 ◽  
Vol 5 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Ellicott McConnell ◽  
A. Glenn Richards
Keyword(s):  
Bacillus Thuringiensis ◽  
Inclusion Bodies ◽  
Galleria Mellonella ◽  
Culture Media ◽  
Water Soluble ◽  
Broth Culture ◽  
Toxic Principle ◽  
Heat Stable

Bacillus thuringiensis Berliner produces in vitro a heat-stable, dialyzable substance which is toxic for insects when injected. The same or a similar substance is produced in vivo. The toxic principle is of unknown composition. It is heat-stable, water-soluble, dialyzable, and resistant to low temperatures. It is probably neither a protein nor a lipid. Clearly it is distinct from the heat-labile inclusion bodies and from lecithinase. Growth-curve studies showed that the heat-stable toxin appeared in liver broth cultures during the active growth phase, prior to the formation of spores or inclusion bodies. An attempt to produce the toxic principle from culture media in the absence of bacteria was unsuccessful from sterile inocula both from in vivo and in vitro sources. The LD50 for larvae of Galleria mellonella injected with autoclaved supernatant from a 10-day-old liver broth culture of B. thuringiensis was determined to be 0.00036 ml per larva or 0.002 ml per gram of larvae. Approximately the same level of toxicity was found for another caterpillar, a fly larva, and cockroaches. After larvae of Galleria or Pyrausla have been dead for more than 2 days from infection with B. thuringiensis the bacillus could no longer be recovered. A sublethal amount of the heat-stable toxin injected into old larvae of Galleria delayed emergence of the adults by 30 to 40%. The non-pathogenic Bacillus cereus was found to produce a similar-acting, heat-stable toxin under the same conditions that one is produced by B. thuringiensis.

scholarly journals Function of the N-terminal propeptide of an aminopeptidase from Vibrio proteolyticus

2000 ◽  
Vol 350 (3) ◽  
pp. 671-676 ◽  
Author(s):  
Zhen-Zhong ZHANG ◽  
Satoru NIRASAWA ◽  
Yoshiaki NAKAJIMA ◽  
Michiteru YOSHIDA ◽  
Kiyoshi HAYASHI
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Active Region ◽  
Protein Expression ◽  
Inclusion Bodies ◽  
Active Enzyme ◽  
Vibrio Proteolyticus ◽  
Show Evidence ◽  
Inhibited Enzyme

An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli. Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro. Using l-Leu-p-nitroanilide as the substrate, the kinetic parameters (kcat and Km) of the pro-aminopeptidase and processed aminopeptidases were analysed. The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region. In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region.

scholarly journals Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

2005 ◽  
Vol 79 (15) ◽  
pp. 10023-10031 ◽  
Author(s):  
Jens Modrof ◽  
Kostas Lymperopoulos ◽  
Polly Roy
Keyword(s):  
Bluetongue Virus ◽  
Inclusion Bodies ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Activity ◽  
Nonstructural Protein 2 ◽  
The Matrix ◽  
Infected Cells ◽  
Viral Inclusion

ABSTRACT In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.

scholarly journals Preliminary analysis of PGRP-LC gene and structure characteristics in bumblebees

Sociobiology ◽  
2019 ◽  
Vol 66 (2) ◽  
pp. 348 ◽  
Author(s):  
Minming Chen ◽  
Nanhui Ye ◽  
Yanjie Liu ◽  
Jiandong An
Keyword(s):  
Disulfide Bonds ◽  
Inclusion Bodies ◽  
Coi Gene ◽  
Binding Characteristics ◽  
High Conservation ◽  
Binding Residues ◽  
Binding Groove ◽  
First Time ◽  
Recognition Receptor

PGRP-LC is a significant pattern recognition receptor of the insect innate immune system that can recognize peptidoglycans and activate immune signaling pathways regulating the expression and release of antimicrobial peptides against infection. We for the first time analyzed the phylogenetic tree, purification and structure of bumblebee PGRP-LC. The results showed high conservation of bumblebee PGRP-LC among the 16 bumblebee species, and further phylogenetic analysis showed that the PGRP-LC phylogeny of different subgenera (Subterraneobombus, Megabombus, Melanobombus, Bombus) is consistent with that of the COI gene. Additionally, the phylogeny of PGRP-LCs among Bombus, Apis and the solitary bee Megachile rotundata coincides with the sociality evolution of bees. Moreover, bumblebee PGRP-LC (Bl-PGRP-LC) shares the Drosophila PGRP-LCx and PGRP-LCa topology, retaining conserved disulfide bonds and 80% binding residues involved in the interaction between TCT and PGRP-LCx. Therefore, Bl-PGRP-LC might share some similar binding characteristics with Drosophila PGRP-LCx. In addition, Bl-PGRP-LC has shorter β5 and β1 sheets, longer β2, β3, and β4 sheets and a shallow binding groove. To determine the characteristics of Bl-PGRP-LC, high-purity PGRP-LC inclusion bodies, soluble GST-tag Bl-PGRP-LC fusion protein and soluble pure Bl-PGRP-LC were obtained in vitro. The results will be helpful for further study of the function and structure of Bl-PGRP-LC.

scholarly journals Detection and characterization of proteins encoded by the second ORF of the M2 gene of pneumoviruses

1999 ◽  
Vol 80 (8) ◽  
pp. 2011-2016 ◽  
Author(s):  
G. Ahmadian ◽  
P. Chambers ◽  
A. J. Easton
Keyword(s):  
Inclusion Bodies ◽  
Virus Protein ◽  
Sequence Identity ◽  
Avian Pneumovirus ◽  
Orf2 Protein ◽  
Monospecific Antisera ◽  
Gst Fusion Proteins

The nucleotide sequence of the M2 gene of pneumonia virus of mice (PVM) was determined. The sequence showed that the gene encoded a protein of 176 amino acids with a predicted molecular mass of 20165 Da from a major ORF, which is smaller than the equivalent proteins encoded by human, bovine and ovine respiratory syncytial (RS) viruses. The PVM M2 protein is conserved, having 41% similarity to the equivalent human RS virus protein. In common with the M2 genes of the RS viruses and avian pneumovirus (APV), the PVM mRNA also contained a second ORF (ORF2) that partially overlaps the first ORF and which is capable of encoding a 98 residue polypeptide. No significant sequence identity could be detected between the putative M2 ORF2 proteins of PVM, APV and the RS viruses. The expression of the M2 ORF2 proteins of the pneumoviruses was investigated by using monospecific antisera raised against GST fusion proteins. Western blot analysis demonstrated the presence of polypeptides encoded by M2 ORF2 of PVM and RS virus corresponding with those predicted by in vitro translation studies, but this was not the case for APV. The PVM polypeptide was present as three distinct products in vivo. The PVM and RS virus polypeptides were also detected in cells by immunofluorescence, which showed that both were present in the cytoplasm with a degree of localization in inclusion bodies. No APV M2 ORF2 protein could be detected in vivo. The RS virus M2 ORF2 polypeptide was shown to accumulate during infection and the potential implications of this are discussed.

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