Comparing the efficiency of different Escherichia coli strains in producing recombinant capsid protein of porcine circovirus type 2

Rai, Vishal; Upmanyu, Vikramaditya; Mohd, Gulam; Kumar, Ravi; Koppad, Sanganagouda; Ansari, Aleema; Bora, Durlav Prasad; Pandey, Awadh Bihari; Dhar, P.; Tiwari, Ashok Kumar

doi:10.1016/j.mcp.2020.101556

Cited by 9 publications

(3 citation statements)

References 18 publications

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“…The E. coli BL21 (DE3) was temporarily suspended and considered to be unable to express RgPAL due to codon bias problems. Rosetta TM host strains, as BL21 derivatives, are designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli (Yin et al, 2007;Rai et al, 2020). Subsequently, we changed the host to E. coli Rosetta-Gami 2 (DE3) with an abundance of tRNAs for rare codons and expressed the protein under different induction conditions with respect to RgPAL.…”

Section: Results Of Protein Expression and Codon Optimizationmentioning

confidence: 99%

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

Xue

Liu

et al. 2021

Front. Bioeng. Biotechnol.

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PAL (phenylalanine ammonia lyase) is important for secondary metabolite production in plants and microorganisms. There is broad interest in engineering PAL for its biocatalytic applications in industry, agriculture, and medicine. The production of quantities of high-activity enzymes has been explored by gene cloning and heterogeneous expression of the corresponding protein. Here, we cloned the cDNA of Rhodotorula glutinis PAL (RgPAL) and introduced codon optimization to improve protein expression in Escherichia coli and enzyme activities in vitro. The RgPAL gene was cloned by reverse transcription and named pal-wt. It had a full-length of 2,121 bp and encoded a 706-amino-acid protein. The pal-wt was inefficiently expressed in E. coli, even when the expression host and physical conditions were optimized. Therefore, codon optimization was used to obtain the corresponding gene sequence, named pal-opt, in order to encode the same amino acid for the RgPAL protein. The recombinant protein encoded by pal-opt, named PAL-opt, was successfully expressed in E. coli and then purified to detect its enzymatic activity in vitro. Consequently, 55.33 ± 0.88 mg/L of PAL-opt protein with a specific activity of 1,219 ± 147 U/mg and Km value of 609 μM for substrate L-phenylalanine was easily obtained. The enzyme protein also displayed tyrosine ammonia lyase (TAL)–specific activity of 80 ± 2 U/mg and Km value of 13.3 μM for substrate L-tyrosine. The bifunctional enzyme RgPAL/TAL (PAL-opt) and its easy expression advantage will provide an important basis for further applications.

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Section: Results Of Protein Expression and Codon Optimizationmentioning

confidence: 99%

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

Xue

Liu

et al. 2021

Front. Bioeng. Biotechnol.

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“…Choosing a proper host is very important in biotechnology. Due to its diverse molecular networks, each species exhibits different behaviors when expressing recombinant proteins (de Moura et al 2020;Li and Huang 2018;Rai et al 2020). The most frequent prokaryotic strain which has been very widely used to express recombinant proteins is E. coli BL21 (DE3) (Makino et al 2011;Rosano and Ceccarelli 2014).…”

Section: Discussionmentioning

confidence: 99%

WITHDRAWN: Evaluating the Five Escherichia coli Derivative Strains as Platform for Arginine Deiminase Overproduction

Abdollahi

Morowvat

Savardashtaki

et al. 2020

Preprint

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Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. This study attempted to evaluate the five host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHuffle in terms of arginine deiminase (ADI) production and enzyme activity. Arginine deiminase (ADI) was chosen a bacterial enzyme which degrades L-arginine. It is effective in treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC) which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through CaCl 2 method. It was then transformed with the construct of pET3a-ADI using heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

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“…Diagnostic testing of the PCV2 capsid is useful for monitoring infection and helps swine farmers to assess whether there are subpopulations and implement strategies to homogenize the immune status of the breeding herd. Many tools are available for detection of PCV2 including quantitative polymerase chain reaction (PCR) that is widely used to detect PCV2 in serum samples, immunoperoxidase monolayer assay (IPMA), western blotting, enzyme-linked immunosorbent assay (ELISA), , and surface plasmon resonance (SPR) . These methods require antibodies specific to the PCV2 capsid protein.…”

Section: Introductionmentioning

confidence: 99%

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development

et al. 2021

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Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD) that causes huge global economic losses for the swine industry. Effective strategies or rapid detection of PCV2 in pig are essential to control PCVAD. Here, single-chain variable fragments (scFvs) were selected and characterized against the PCV2 capsid using phage display technology. Phage scFv clones were selected from the human scFv phagemid library (Tomlinson I + J) for direct panning against the PCV2 capsid. Eighty-four monoclonal phage scFvs were individually tested for binding to the PCV2 capsid by ELISA. Eight scFv clones showed significant binding to the PCV2 capsid and only three clones (clone nos. 13, 37, and 81) contained both VHCDRs and VLCDRs in the sequence. Clone scFv no. 81 had the highest reactivity to the PCV2 capsid and was constructed in the pET22b (+) expression vector. The recombinant was transformed to Escherichia coli BL21(DE3) for expression and purification. The scFv showed appropriate affinity to the PCV2 capsid by western blot analysis. Kinetics of scFv and the PCV2 capsid were determined using surface plasmon resonance and showed binding affinity in the nanomolar range (K D = 57.2 nM). Our scFv was first applied in the development of an impedimetric immunosensor for PCV2 capsid detection, and results showed that impedance increased with increasing PCV2 capsid expression with limit of detection = 114 nM. Findings demonstrated that our scFv has potential for use as a receptor for biosensor devices.

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Comparing the efficiency of different Escherichia coli strains in producing recombinant capsid protein of porcine circovirus type 2

Cited by 9 publications

References 18 publications

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

WITHDRAWN: Evaluating the Five Escherichia coli Derivative Strains as Platform for Arginine Deiminase Overproduction

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development

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