scholarly journals Codon optimization of Col H gene encoding Clostridium histolyticum collagenase to express in Escherichia coli

2014 ◽  
Author(s):  
Hamzeh Alipour ◽  
Abbasali Raz ◽  
Navid Dinparast Djadid ◽  
Abbas Rami ◽  
Seyed Mohammad Amin Mahdian
Keyword(s):  
Escherichia Coli ◽  
Codon Usage ◽  
Codon Optimization ◽  
Coding Region ◽  
Gene Encoding ◽  
E Coli ◽  
Clostridium Histolyticum ◽  
Optimal Codons ◽  
H Gene ◽  
Clostridium Histolyticum Collagenase

A given amino acid sequence can be encoded by a huge number of different nucleic acid sequences. These sequences, however, prove not to be equally useful. The choice of sequence can significantly impact the expression of an encoded protein. As regards the importance of protein-coding sequence and promising industrial and medicinal applications of Clostridium histolyticum collagenase, this study examined the codon optimization of the Col H gene so as to enhance collagenase expression in Escherichia coli (E. coli). The coding region of mature Col H gene was optimized according to the codon usage of E. coli using Gene Designer software (DNA 2.0). The results revealed that relative frequency of codon usage in Col H gene was adapted to the most preferred triplets in E. coli in such a way that codon usage bias in E. coli was enhanced after codon optimization. Similarly, the higher level of collagenase expression was more likely the result of substituting rare codons with optimal codons. As has been reported elsewhere, the findings from this study suggest that codon optimization provides a theoretical improvement in Col H gene expression in E. coli. In spite of that, experimental research is needed to confirm the improvement.

scholarly journals Codon optimization of Col H gene encoding Clostridium histolyticum collagenase to express in Escherichia coli

2014 ◽  
Author(s):  
Hamzeh Alipour ◽  
Abbasali Raz ◽  
Navid Dinparast Djadid ◽  
Abbas Rami ◽  
Seyed Mohammad Amin Mahdian
Keyword(s):  
Escherichia Coli ◽  
Codon Usage ◽  
Codon Optimization ◽  
Coding Region ◽  
Gene Encoding ◽  
E Coli ◽  
Clostridium Histolyticum ◽  
Optimal Codons ◽  
H Gene ◽  
Clostridium Histolyticum Collagenase

A given amino acid sequence can be encoded by a huge number of different nucleic acid sequences. These sequences, however, prove not to be equally useful. The choice of sequence can significantly impact the expression of an encoded protein. As regards the importance of protein-coding sequence and promising industrial and medicinal applications of Clostridium histolyticum collagenase, this study examined the codon optimization of the Col H gene so as to enhance collagenase expression in Escherichia coli (E. coli). The coding region of mature Col H gene was optimized according to the codon usage of E. coli using Gene Designer software (DNA 2.0). The results revealed that relative frequency of codon usage in Col H gene was adapted to the most preferred triplets in E. coli in such a way that codon usage bias in E. coli was enhanced after codon optimization. Similarly, the higher level of collagenase expression was more likely the result of substituting rare codons with optimal codons. As has been reported elsewhere, the findings from this study suggest that codon optimization provides a theoretical improvement in Col H gene expression in E. coli. In spite of that, experimental research is needed to confirm the improvement.

scholarly journals Strategy for Designing the Synthetic Gene Encoding Human papillomavirus Major Capsid L1 Protein for Heterologous Expression in Escherichia coli System

2020 ◽  
Vol 8 (2) ◽  
pp. 225
Author(s):  
Kartika Sari Dewi ◽  
Wien Kusharyoto
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Codon Optimization ◽  
Gc Content ◽  
Web Server ◽  
Synthetic Gene ◽  
Gene Encoding ◽  
E Coli ◽  
Gene Construct ◽  
L1 Protein

DNA is widely used to construct heterologously expressed genes. The adaptation of the codons to the host organism is necessary in order to ensure sufficient production of proteins. The GC content, codon identity and the mRNA from the translation site are also important in the design of the gene construct. This study performed a strategy for the design of synthetic gene encoding HPV52 L1 protein and several analyses at the genetic level to optimize its protein expression in the Escherichia coli BL21(DE3) host. The determination of the codon optimization was performed by collecting 75 HPV52 L1 protein sequences in the NCBI database. Furthermore, all the sequences were analyzed using multiple global alignments by Clustal Omega web server. Once the model was determined, codon optimization was performed using OPTIMIZER and the web server of the IDT codon optimization tool based on the E. Coli B. The generated open reading frame (ORF) sequence was analyzed using Restriction mapper web server to choose the restriction site for facilitating the cloning stage, which is adjusted for pJExpress414 expression vector. To maximize the protein expression level, the mRNA secondary structure analysis around the ribosome binding site (rbs) was performed. A slight modification at the 5’-terminal end waa carried out in order to get more accessible rbs and increasing mRNA folding free energy. Finally, the construction of the synthetic gene was confirmed to ensure that no mutation occurs in the protein and to calculate its Codon Adaptation Index (CAI) and GC content. The above strategy, which leads to a good ORF sequence with the value of the free mRNA folding energy around rbs, is -5.5 kcal / mol, CAI = 0.787 and GC content 49.5%. This result is much better than its original gene. This result is much better compared to its native gene. Theoretically it is possible that this synthetic gene construct generates a high level protein expression in E. coli BL21 (DE3) under the regulation of the T7 promoter.

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1421-1431 ◽  
Author(s):  
Patrice Bruscella ◽  
Laure Cassagnaud ◽  
Jeanine Ratouchniak ◽  
Gaël Brasseur ◽  
Elisabeth Lojou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acidithiobacillus Ferrooxidans ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Tat System ◽  
Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

Increased Production of Recombinant O-Phospho-L-Serine Sulfhydrylase from the Hyperthermophilic Archaeon Aeropyrum pernix K1 Using Escherichia coli

2019 ◽  
Vol 8 (1) ◽  
pp. 15-23
Author(s):  
Takashi Nakamura ◽  
Emi Takeda ◽  
Tomoko Kiryu ◽  
Kentaro Mori ◽  
Miyu Ohori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Industrial Production ◽  
Codon Optimization ◽  
Scale Production ◽  
Enzymatic Production ◽  
Hyperthermophilic Archaeon ◽  
E Coli ◽  
Aeropyrum Pernix ◽  
Natural Amino Acids

Background: O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce nonnatural amino acids in addition to L-cysteine. Objective: We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for the convenience of research and for industrial production of L-cysteine and non-natural amino acids. Method: We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield. Results and Discussion: E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production. The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS. Conclusion: We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, an enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.

scholarly journals Iha: a Novel Escherichia coli O157:H7 Adherence-Conferring Molecule Encoded on a Recently Acquired Chromosomal Island of Conserved Structure

2000 ◽  
Vol 68 (3) ◽  
pp. 1400-1407 ◽  
Author(s):  
Phillip I. Tarr ◽  
Sima S. Bilge ◽  
James C. Vary ◽  
Srdjan Jelacic ◽  
Rebecca L. Habeeb ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Vibrio Cholerae ◽  
Shiga Toxin ◽  
Escherichia Coli O157 ◽  
Chromosomal Gene ◽  
Gene Encoding ◽  
Tellurite Resistance ◽  
E Coli ◽  
Dna Library

ABSTRACT The mechanisms used by Shiga toxin (Stx)-producingEscherichia coli to adhere to epithelial cells are incompletely understood. Two cosmids from an E. coliO157:H7 DNA library contain an adherence-conferring chromosomal gene encoding a protein similar to iron-regulated gene A (IrgA) ofVibrio cholerae (M. B. Goldberg, S. A. Boyko, J. R. Butterton, J. A. Stoebner, S. M. Payne, and S. B. Calderwood, Mol. Microbiol. 6:2407–2418, 1992). We have termed the product of this gene the IrgA homologue adhesin (Iha), which is encoded by iha. Iha is 67 kDa in E. coliO157:H7 and 78 kDa in laboratory E. coli and is structurally unlike other known adhesins. DNA adjacent toiha contains tellurite resistance loci and is conserved in structure in distantly related pathogenic E. coli, but it is absent from nontoxigenic E. coli O55:H7, sorbitol-fermenting Stx-producing E. coli O157:H−, and laboratory E. coli. We have termed this region the tellurite resistance- and adherence-conferring island. We conclude that Iha is a novel bacterial adherence-conferring protein and is contained within an E. coli chromosomal island of conserved structure. Pathogenic E. coli O157:H7 has only recently acquired this island.

scholarly journals Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

2011 ◽  
Vol 77 (20) ◽  
pp. 7104-7112 ◽  
Author(s):  
Maria Karczmarczyk ◽  
Yvonne Abbott ◽  
Ciara Walsh ◽  
Nola Leonard ◽  
Séamus Fanning
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Gene Array ◽  
Multidrug Resistant ◽  
Genetic Determinants ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

scholarly journals Reconstitution of Acetosyringone-Mediated Agrobacterium tumefaciens Virulence Gene Expression in the Heterologous Host Escherichia coli

2001 ◽  
Vol 183 (12) ◽  
pp. 3704-3711 ◽  
Author(s):  
Scott M. Lohrke ◽  
Hongjiang Yang ◽  
Shouguang Jin
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Virulence Gene Expression ◽  
E Coli ◽  
Glucose Binding

ABSTRACT The ability to utilize Escherichia coli as a heterologous system in which to study the regulation ofAgrobacterium tumefaciens virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the rpoA gene encoding the alpha subunit of RNA polymerase is required for the expression of lacZ gene under the control of virB promoter (virBp::lacZ) in E. colicontaining a constitutively active virG gene [virG(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from E. coli and the C-terminal 89 residues from A. tumefaciens was able to significantly express virBp::lacZ in E. coli in a VirG(Con)-dependent manner. Utilization oflac promoter-driven virA and virGin combination with the A. tumefaciens rpoA construct resulted in significant inducer-mediated expression of thevirBp::lacZ fusion, and the level ofvirBp::lacZ expression was positively correlated to the copy number of the rpoA construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in A. tumefaciens. Furthermore, the effect of sugars on virgene expression was observed only in the presence of thechvE gene, suggesting that the glucose-binding protein ofE. coli, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in A. tumefaciens strain A348 from which the virA clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of thevir genes in E. coli is less than what is observed in A. tumefaciens, suggesting that additional gene(s) from A. tumefaciens may be required for the full expression of virulence genes in E. coli.

scholarly journals Variations in O-Antigen Biosynthesis and O-Acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s

2014 ◽  
Vol 197 (5) ◽  
pp. 905-912 ◽  
Author(s):  
Yuriy A. Knirel ◽  
Nikolai S. Prokhorov ◽  
Alexander S. Shashkov ◽  
Olga G. Ovchinnikova ◽  
Evelina L. Zdorovenko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cells ◽  
Side Chain ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Content Type ◽  
Surface Receptors ◽  
E Coli ◽  
O Antigen ◽  
Phage Sensitivity

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmentalEscherichia coliisolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen ofE. coliO22, differing only in side-chain α-d-glucosylation in the former, mediated by agtrlocus on the chromosome. Spontaneous mutations ofE. coli4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions ofE. coli4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.

A novel peroxiredoxin of the plant Sedum lineare is a homologue of Escherichia coli bacterioferritin co-migratory protein (Bcp)

2000 ◽  
Vol 351 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Wei KONG ◽  
Susumu SHIOTA ◽  
Yixin SHI ◽  
Hiroaki NAKAYAMA ◽  
Koji NAKAYAMA
Keyword(s):  
Escherichia Coli ◽  
Peroxidase Activity ◽  
Discrete Group ◽  
Sequence Data ◽  
Accession Number ◽  
Gene Encoding ◽  
Reaction Cycle ◽  
Mutant Proteins ◽  
E Coli

We cloned a gene encoding a 17-kDa protein from a cDNA library of the plant Sedum lineare and found that its deduced amino acid sequence showed similarities to those of Escherichia coli bacterioferritin co-migratory protein (Bcp) and its homologues, which comprise a discrete group associated with the peroxiredoxin (Prx) family. Studies of the recombinant 17-kDa protein produced in E. coli cells revealed that it actually had a thioredoxin-dependent peroxidase activity, the hallmark of the Prx family. PrxQ, as we now designate the 17-kDa protein, had two cysteine residues (Cys-44 and Cys-49) well conserved among proteins of the Bcp group. These two cysteines were demonstrated to be essential for the thioredoxin-dependent peroxidase activity by analysis of mutant proteins, suggesting that these residues are involved in the formation of an intramolecular disulphide bond as an intermediate in the reaction cycle. Expression of PrxQ suppressed the hypersensitivity of an E. coli bcp mutant to peroxides, indicating that it might exert an antioxidant activity in vivo. The sequence data presented have been deposited in the GenBank/EMBL/DDBJ nucleotide sequence databases under the accession number AB037598.

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis

1992 ◽  
Vol 38 (4) ◽  
pp. 350-353 ◽  
Author(s):  
A. Moreau ◽  
F. W. Paradis ◽  
R. Morosoli ◽  
F. Shareck ◽  
D. Kluepfel
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Shuttle Vector ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Single Stranded Dna ◽  
E Coli ◽  
Streptomyces Spp ◽  
Ampicillin Resistance Gene

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective markerin E. coli. Vector pIAFS20 also contains the phage fl intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screeningof the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis. Key words: shuttle vector, single-stranded DNA, site-directed mutagenesis, Streptomyces spp., Escherichia coli.

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