scholarly journals Isolation and characterization of recombinant Drosophila Copia aspartic proteinase

2006 ◽  
Vol 399 (3) ◽  
pp. 535-542 ◽  
Author(s):  
Senarath B. P. Athauda ◽  
Katsuji Yoshioka ◽  
Tadayoshi Shiba ◽  
Kenji Takahashi
Keyword(s):  
Active Site ◽  
Cleavage Site ◽  
Bovine Insulin ◽  
Precursor Protein ◽  
Coding Region ◽  
Aspartic Proteinases ◽  
E Coli ◽  
Isolation And Characterization ◽  
Nacl Concentration ◽  
Molecular Masses

The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe–Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 °C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15−–Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.

Cloning and optimizing the expression of the DHDPS gene in the Medicago truncatula

2019 ◽  
Author(s):  
Hoang Thi Kim Hong ◽  
Pham Thi Hong Trang ◽  
Dang Thanh Long ◽  
Nguyen Thi Quynh Trang
Keyword(s):  
Escherichia Coli ◽  
Medicago Truncatula ◽  
Culture Conditions ◽  
Coding Region ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Ncbi Accession ◽  
Molecular Masses ◽  
Induction Times ◽  
Topo Vector

Medicago truncatula seeds were cultured and developed in Thua Thien Hue province, Vietnam, and they were used as materials for cloning a DHDPS gene with the encoding of the isozyme dihydrodipicolinate synthase (DHDPS) as well as optimizing the culture conditions for having the highest DHDPS gene expression in Escherichia coli BL21 StarTM (DE3) cells. The results revealed that the coding region of the DHDPS gene from M. truncatula was 100% similar with M. truncatula 4-hydroxy-tetrahydrodipicolinate synthase 2 (DHDPS2) submitted in NCBI (accession number: XM_013589555.2), coding for a long polypeptide of 307 amino acid with the molecular mass of about 33495 Da (Protein ID: XP_013445009.1). The DHDPS gene was successfully expressed in the Escherichia coli BL21 StarTM (DE3) cells with a pET200 / D-TOPO vector, and this produced the DHDPS2 protein with molecular masses of approximately 33.87 kDa (»33.5 kDa of DHDPS2 and 3.7 kDa of fusion fragment of pET 200/D-TOPO vector). The effects of the six different culture mediums of LB, SOB, SOC, YJ, HSG and TB, the induction times of 2h, 4h, 6h, 8h, 10h and 12h, and the inducer concentrations of 0.2 mM; 0.5 mM; 0.7 mM; 1.0 mM; 1.2 mM and 1.5 mM IPTG (Isopropyl â-D-1-thiogalactopyranoside) were also investigated for the purpose of optimising the expression of DHDPS2 in E. coli cells, and it was found that strong expression of recombinant DHDPS2 protein in E. coli. BL21 (DE3) cells occurred on the TB, HSG and YJ culture mediums after 8 hours with 0.2 mM inducible IPTG (BioRad).

scholarly journals A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast

Genetics ◽  
1987 ◽  
Vol 117 (1) ◽  
pp. 5-12
Author(s):  
Eric Alani ◽  
Nancy Kleckner
Keyword(s):  
Gene Fusion ◽  
Enzyme Assay ◽  
Decarboxylase Activity ◽  
Lacz Gene ◽  
Coding Region ◽  
Ura3 Gene ◽  
Promoter Sequences ◽  
E Coli ◽  
New Type ◽  
Fusion Analysis

ABSTRACT We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5′-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.

scholarly journals A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site.

1991 ◽  
Vol 88 (23) ◽  
pp. 10792-10796 ◽  
Author(s):  
A. R. Welch ◽  
A. S. Woods ◽  
L. M. McNally ◽  
R. J. Cotter ◽  
W. Gibson
Keyword(s):  
Active Site ◽  
Cleavage Site ◽  
Putative Active Site ◽  
Its Gene

Forebrain and midbrain regions are deleted in Otx2−/− mutants due to a defective anterior neuroectoderm specification during gastrulation

Development ◽  
1995 ◽  
Vol 121 (10) ◽  
pp. 3279-3290 ◽  
Author(s):  
D. Acampora ◽  
S. Mazan ◽  
Y. Lallemand ◽  
V. Avantaggiato ◽  
M. Maury ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Null Allele ◽  
External Layer ◽  
Coding Region ◽  
Coding Sequence ◽  
E Coli ◽  
Homozygous Mutant

We have replaced part of the mouse homeogene Otx2 coding region with the E. coli lacZ coding sequence, thus creating a null allele of Otx2. By 9.5 dpc, homozygous mutant embryos are characterized by the absence of forebrain and midbrain regions. From the early to midstreak stages, endomesodermal cells expressing lacZ fail to be properly localized anteriorly. In the ectodermal layer, lacZ transcription is progressively extinguished, being barely detectable by the late streak stage. These data suggest that Otx2 expression in endomesoderm and ectoderm is required for anterior neuroectoderm specification. In gastrulating heterozygous embryos, a post-transcriptional repression acts on lacZ transcripts in the ectoderm, but not in the external layer, suggesting that different post-transcriptional mechanisms control Otx2 expression in both layers.

scholarly journals Long-range RNA interaction of two sequence elements required for endonucleolytic cleavage of human insulin-like growth factor II mRNAs.

1995 ◽  
Vol 15 (1) ◽  
pp. 235-245 ◽  
Author(s):  
W Scheper ◽  
D Meinsma ◽  
P E Holthuizen ◽  
J S Sussenbach
Keyword(s):  
Growth Factor ◽  
Long Range ◽  
Cleavage Site ◽  
Untranslated Region ◽  
Cleavage Product ◽  
Coding Region ◽  
Endonucleolytic Cleavage ◽  
Factor Ii ◽  
Sequence Elements ◽  
Rna Interaction

Human insulin-like growth factor II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3' untranslated region, leading to an unstable 5' cleavage product containing the IGF-II coding region and a very stable 3' cleavage product of 1.8 kb. This endonucleolytic cleavage is most probably the first and rate-limiting step in degradation of IGF-II mRNAs. Two sequence elements within the 3' untranslated region are required for cleavage: element I, located approximately 2 kb upstream of the cleavage site, and element II, encompassing the cleavage site itself. We have identified a stable double-stranded RNA stem structure (delta G = -100 kcal/mol [418.4 kJ/mol]) that can be formed between element I and a region downstream of the cleavage site in element II. This structure is conserved among human, rat, and mouse mRNAs. Detailed analysis of the requirements for cleavage shows that the relative position of the elements is not essential for cleavage. Furthermore, the distance between the coding region and the cleavage site does not affect the cleavage reaction. Mutational analysis of the long-range RNA-RNA interaction shows that not only the double-stranded character but also the sequence of the stable RNA stem is important for cleavage.

scholarly journals Isolation and characterization of escherichia coli in ready-to-eat foods vended in Islamic University, Kushtia

1970 ◽  
Vol 18 ◽  
pp. 99-103 ◽  
Author(s):  
S Biswas ◽  
MAK Parvez ◽  
M Shafiquzzaman ◽  
S Nahar ◽  
MN Rahman
Keyword(s):  
Escherichia Coli ◽  
Pattern Analysis ◽  
University Campus ◽  
Rflp Pattern ◽  
E Coli ◽  
Fish Meat ◽  
Isolation And Characterization ◽  
Food Borne ◽  
Identification And Characterization

Context: Escherichia coli is shed in the feces of warm blooded animals and humans and thus potential for public health. Detection and characterization of E. coli in the ready-to-eat (RTE) foods concerns due to their presence indicates fecal contamination of the food.   Objective: To identify, characterize and RFLP pattern analysis of E. coli isolated from RTE foods vended in Islamic University campus, Kushtia.   Materials and Methods: Fifty samples from four types of consumed foods in six student halls of residence, some temporary restaurants of Islamic University, Kushtia were assessed for bacterial contamination by standard methods. Identification and characterization of E. coli isolates were performed using IMViC tests. Genomic DNA was used to perform RFLP pattern analysis.   Results: Thirty seven out of 50 (74%) examined samples of RTE foods had E. coli contamination. The highest number of E. coli was isolated from vegetable oriented RTE foods (90.90%) and fish, meat and cereals samples were also significantly E. coli positive. RFLP profiling of two E. coli isolates were observed.   Conclusion: The results of this study provide evidence that some RTE foods had unsatisfactory levels of contamination with E. coli. Thus street vended RTE food could be important potential vehicles for food-borne diseases. Molecular characterization may be exploited to identify food borne pathogen among different species.  Keywords: Ready-to-eat foods; Escherichia coli; RFLP pattern DOI: http://dx.doi.org/10.3329/jbs.v18i0.8783 JBS 2010; 18(0): 99-103

scholarly journals Molecular Characterization of Mycoplasma arthritidis Variable Surface Protein MAA2

1998 ◽  
Vol 66 (6) ◽  
pp. 2576-2586 ◽  
Author(s):  
Leigh Rice Washburn ◽  
Keith E. Weaver ◽  
Elizabeth J. Weaver ◽  
Wendy Donelan ◽  
Suhaila Al-Sheboul
Keyword(s):  
Amino Acid ◽  
Signal Peptide ◽  
Dna Sequences ◽  
Surface Protein ◽  
Carboxypeptidase Y ◽  
Coding Region ◽  
Intact Cells ◽  
Mycoplasma Arthritidis ◽  
E Coli ◽  
Variable Surface

Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability. Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M. arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2. Triton X-114 partitioning and metabolic labeling with [3H]palmitic acid suggested lipid modification of MAA2. Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y. The maa2genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats. The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide. The maa2 gene was expressed inEscherichia coli from the lacZ promoter of vector pGEM-T. The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E. coli. The maa2 gene and upstream DNA sequences were cloned from M. arthritidisclonal variants differing in MAA2 expression state. Expression state correlated with the length of a poly(T) tract just upstream of a putative −10 box. Full-sized recombinant MAA2 was expressed inE. coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.

scholarly journals Enzymes and Substrates Are Balanced at Minimal Combined Mass Concentration in vivo

2017 ◽  
Author(s):  
Hugo Dourado ◽  
Veronica G. Maurino ◽  
Martin J. Lercher
Keyword(s):  
Fundamental Problem ◽  
Geometric Mean ◽  
Physiological State ◽  
Cellular Systems ◽  
Cellular Growth ◽  
Metabolic Efficiency ◽  
E Coli ◽  
Molecular Masses ◽  
Enzyme Molecules

AbstractA fundamental problem in biology is how cells organize their resource investment. Cellular metabolism, for example, typically involves hundreds of enzymes and metabolites, but it is unclear according to which principles their concentrations are set. Reasoning that natural selection will drive cells towards achieving a given physiological state at minimal cost, we derive a general equation that predicts the concentration of a metabolite from the concentration of the most abundant and costly enzyme consuming it. Simulations of cellular growth as well as experimental data demonstrate that costs are approximately proportional to molecular masses. For effectively irreversible reactions, the cell maximizes its metabolic efficiency by investing equally into substrate and unbound enzyme molecules. Without fitting any free parameters, the resulting model predicts in vivo substrate concentrations from enzyme concentrations and substrate affinities with high accuracy across data from E. coli and diverse eukaryotes (R2=0.79, geometric mean fold-error 1.74). The corresponding organizing principle – the minimization of the summed mass concentrations of solutes – may facilitate reducing the complexity of kinetic models and will contribute to the design of more efficient synthetic cellular systems.

scholarly journals Isolation and characterization of bacteria residing in the oral, gut, and fecal samples of different pheasant species

2023 ◽  
Vol 83 ◽  
Author(s):  
M. Mushtaq ◽  
S. M. Bukhari ◽  
S. Ahmad ◽  
A. Khattak ◽  
M. B. Chattha ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Gut Contents ◽  
Phasianus Colchicus ◽  
Gut Content ◽  
E Coli ◽  
Colony Forming Unit ◽  
Isolation And Characterization ◽  
Staphylococcus Spp ◽  
Campylobacter Spp

Abstract There is a paucity of research conducted on microbial prevalence in pheasants. The microbiota of captive birds has zoonotic significance and must be characterize. Present study is therefore planned to assess the microbiota from oral, fecal and gut content of captive avian species. It will be helpful in characterization of harmful microbes. Different samples taken from oral, gut and feces of ring-necked pheasants (Phasianus colchicus), green pheasants (Phasianus versicolor), golden pheasant (Chrysolophus pictus) and silver pheasant (Lophura nycthemera). Samples were collected, diluted, and inoculated onto different agar plates (MacConkey, SS agar, MSA and nutrient agar) for cultivation of bacterial species. Colonies of E.coli, Staphylococcus spp. Brachyspira spp. and Campylobacter spp were observed based on colony morphology. Colony forming unit showed E. coli as frequently found bacteria in fecal, oral and gut contents of all the above pheasants. The overall significance difference was found among bacterial species of golden pheasants, green pheasant, ring-necked pheasant, and silver pheasants. It was concluded that E.coli is predominant isolated from heathy pheasants followed by Campylobacter, Staphylococcus and Brachyspira.

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