Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli

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.

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Codon Optimization Enhances Protein Expression of Bombyx mori Nucleopolyhedrovirus DNA Polymerase in E. coli

Current Microbiology ◽

10.1007/s00284-013-0476-5 ◽

2013 ◽

Vol 68 (3) ◽

pp. 293-300 ◽

Cited By ~ 8

Author(s):

Huifang Song ◽

Guohui Li ◽

Weijun Mai ◽

Guoping Huang ◽

Keping Chen ◽

...

Keyword(s):

Protein Expression ◽

Bombyx Mori ◽

Dna Polymerase ◽

Codon Optimization ◽

Bombyx Mori Nucleopolyhedrovirus ◽

E Coli

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

Biogenesis Jurnal Ilmiah Biologi ◽

10.24252/bio.v8i2.15805 ◽

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.

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Cloning of Bacillus subtilis phytase gene construct in Escherichia coli

Iranian Journal of Microbiology ◽

10.18502/ijm.v13i5.7433 ◽

2021 ◽

Author(s):

Mahdiyar Iravani Saadi ◽

Abbas Doosti ◽

Heeva Jalali ◽

Ehsan Nabi Abdolyousefi ◽

Mansooreh Hooshiyar ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Phytic Acid ◽

Restriction Enzymes ◽

Enzymatic Digestion ◽

Bacillus Species ◽

Phytase Gene ◽

Low Phytic Acid ◽

E Coli ◽

Cloning Technique

Background and Objectives: Phytase has a hydrolysis function of phytic acid, which yields inorganic phosphate. Bacillus species can produce thermostable alkaline phytase. The aim of this study was to isolate and clone a Phytase gene (Phy) from Bacillus subtilis in Escherichia coli. Materials and Methods: In this study, the extracellular PhyC gene was isolated from Bacillus subtilis Phytase C. After purification of the bands, DNA fragment of Phy gene was cloned by T/A cloning technique, and the clone was transformed into Escherichia coli. Afterward, the pGEM-Phy was transferred into E. coli Top-10 strain and the recombinants were plated on LB agar containing 100 µg/ml ampicillin. The colonization of 1171 bp of gene Phytase C was confirmed by PCR. The presence of gene-targeting in vector was confirmed with enzymatic digestion by XhoI and XbaI restriction enzymes. Results: The Phytase gene was successfully cloned in E. coli. The result of cloning of 1171 bp Phytase gene was confirmed by PCR assay. Conclusion: Our impression of this article is that several methods, such as using along with microbial, plant phytase reproduction, or low-phytic acid corn may be the better way from a single phytase.

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

10.7287/peerj.preprints.754v1 ◽

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.

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A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture

Applied Microbiology and Biotechnology ◽

10.1007/s00253-016-7683-5 ◽

2016 ◽

Vol 100 (23) ◽

pp. 9979-9994 ◽

Cited By ~ 3

Author(s):

Nirmala Devi ◽

Adivitiya ◽

Yogender Pal Khasa

Keyword(s):

Batch Culture ◽

Codon Optimization ◽

Soluble Expression ◽

Combinatorial Approach ◽

Fed Batch ◽

E Coli ◽

N Terminus ◽

Fed Batch Culture

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Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli

Protein Expression and Purification ◽

10.1016/j.pep.2010.02.011 ◽

2010 ◽

Vol 72 (1) ◽

pp. 101-106 ◽

Cited By ~ 17

Author(s):

Xingxing Wang ◽

Xiujin Li ◽

Zhenlong Zhang ◽

Xinliang Shen ◽

Fei Zhong

Keyword(s):

Pseudomonas Aeruginosa ◽

Codon Optimization ◽

Exotoxin A ◽

Secretory Expression ◽

E Coli ◽

Pseudomonas Aeruginosa Exotoxin

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Codon Preference Optimization Increases Prokaryotic Cystatin C Expression

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/732017 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 6

Author(s):

Qing Wang ◽

Cui Mei ◽

Honghua Zhen ◽

Jess Zhu

Keyword(s):

Protein Expression ◽

Cystatin C ◽

Production Systems ◽

Codon Optimization ◽

Recombinant Protein Expression ◽

Expression System ◽

Gc Content ◽

Optimization Techniques ◽

E Coli ◽

Prokaryotic Expression Vector

Gene expression is closely related to optimal vector-host system pairing in many prokaryotes. Redesign of the humancystatin C(cysC) gene using the preferred codons of the prokaryotic system may significantly increasecysCexpression inEscherichia coli(E. coli). Specifically,cysCexpression may be increased by removing unstable sequences and optimizing GC content. According toE. coliexpression system codon preferences, the gene sequence was optimized while the amino acid sequence was maintained. The codon-optimizedcysC(co-cysC) and wild-typecysC(wt-cysC) were expressed by cloning the genes into a pET-30a plasmid, thus transforming the recombinant plasmid intoE. coliBL21. Before and after the optimization process, the prokaryotic expression vector and host bacteria were examined for protein expression and biological activation of CysC. The recombinant proteins in the lysate of the transformed bacteria were purified using Ni2+-NTA resin. Recombinant protein expression increased from 10% to 46% based on total protein expression after codon optimization. Recombinant CysC purity was above 95%. The significant increase incysCexpression inE. coliexpression produced by codon optimization techniques may be applicable to commercial production systems.

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

10.7287/peerj.preprints.754 ◽

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.

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