Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Young, Carissa L.; Britton, Zachary T.; Robinson, Anne S.

doi:10.1002/biot.201100155

Cited by 388 publications

(265 citation statements)

References 199 publications

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“…Purification is an important step in the efficient production of recombinant protein. PolyHis is the first commercially available affinity tag; it is designed from naturally occurring epitopes and used primarily for purification [77]. Histidine (His) tags are widely used for affinity purification of recombinant proteins in a nickel column [49,78].…”

Section: Peptide Purification and Antimicrobial Activity Evaluationmentioning

confidence: 99%

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

Chahardoli

Fazeli

Niazi‎

et al. 2018

Biotechnology & Biotechnological Equipment

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Antimicrobial peptides (AMPs) with their broad and selective antimicrobial activity and lowered risk of resistance development in microbial populations are promining as a new alternative candidate to conventional antibiotics. Plant-based expression systems can be employed as cost-effective and high scale-up capacity production hosts for recombinant expression of AMPs. LFchimera is a chimerical peptide containing LFcin and LFampin antimicrobial peptides of bovine lactoferrin, and it has stronger bactericidal activity. Here, the LFchimera sequence was codon-optimized for tobacco and fused with endoplasmic reticulum retention signals along with a CaMV 35S promoter and then transferred by agrobacterium-mediated transformation. The integration and expression of the transgene in tobacco plants were confirmed by polymerase chain reaction (PCR) and RT-PCR, respectively. Recombinant production of the peptide was confirmed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), and peptide functional activity was confirmed by the antibacterial evaluation of total protein extracts against various clinical and phytopathogenic bacteria. Finally, LFchimera peptide was partially purified using an affinity column, and the antibacterial activity was shown. Taken together, these results confirmed that tobacco can be utilized for the recombinant production of antimicrobial peptides such as LFchimera.

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Section: Peptide Purification and Antimicrobial Activity Evaluationmentioning

confidence: 99%

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

Chahardoli

Fazeli

Niazi‎

et al. 2018

Biotechnology & Biotechnological Equipment

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“…[6] At the last two decades, the advances in genomics and proteomics made the protein purification more easy for instance, using the affinity tag with the gene of interest can form a fusion gene (therefore, tag is also called a fusion tags) which are then translate to a protein or peptide with a specific properties fused with target protein to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization or analysis of protein structure and function. [7] Examples of present tags are the Histidine, glutathione S-transferase (GST), maltose binding protein (MBP), or Strep-tag TM II. Tags enable recombinant proteins to be purified by affinity chromatography (AC) which is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecules from complex mixtures.…”

Section: Introductionmentioning

confidence: 99%

Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

Al-Badran

Abdul-Jabbar

2017

JBEI

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Objective: The intact human Parathyroid hormone (hPTH) is one of the biopharmaceutical drug produced by biotechnology, this hormone can be provided in a good amount using simple batch culture, and therefore the main purpose of this study was the production of purified bioactive intact hPTH. Methods: A cloning BL21(DE3) strain (which already cloned in our Lab. by synthetic human Parathyroid hormone (shPTH gene) was grown in LB medium and the cloning gene expression was induced by addition of IPTG to the medium. The recombinant fused protein was purified from the bacterial cell lysate by affinity chromatography and underwent proteolysis by Enterokinase enzyme to obtain the target hPTH, and it's further purified by DEAE and SP Sepharose ion exchange chromatography. RP-HPLC analysis and biological activity on the MCF-7 breast cancer cells were done for both the standard and produced hPTH. Furthermore, the haemolysis ability of the latter was tested on the human RBC. Results: 225 ng/ml of hPTH was produced after SP chromatography which detected by specific PTH ELISA kit, standard and produced hPTH showed the same peaks in the same retention time when analyzed by HPLC. The produced hPTH haemolysis assay showed the ability of the produced hPTH to partially haemolyze human RBC in a concentration above 200 µg/ml. The bioactivity assay for the produced and standard rhPTH demonstrated that both compounds had a biological activity on the MCF-7 cells with IC50 of about 84.4 and 75.7 µg/ml for standard and produced hPTH respectively, and no significant difference was detected between them. Conclusions: Via suitable purification processes, the biologically active hPTH with structure similar to the standard ones as detected by RP-HPLC and bioactivity assay, can be obtained by using already cloned isolate with shPTH gene for hormone production. The hormone showed haemolysis effect on the RBC similar to the normal secreted hPTH.

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“…Therefore YHRC potentially could offer a robust methodology for the assembly E. coli expression vectors. Current approaches for recombinant protein purification frequently rely on affinity tags such as the hexahistidine tag (His6), Glutathione--S--transferase or Maltose Binding Protein [18]. Certain tags have the added benefit of enhancing the soluble expression of its fusion partner [19].…”

Section: Introductionmentioning

confidence: 99%

A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination

Holmberg

Gowda

Andréasson

2014

Protein Expression and Purification

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A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.Protein Expression and Purification, http://dx.doi.org/10.1016/j. pep.2014.03.002 Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. Permanent link to this version AbstractProduction of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single--gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET--based bacterial expression vector pSUMO--YHRC. The vector supports cloning for untagged expression as well as fusions to His6--SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single--step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs. Protein expression/Protein purification/cloning/recombination cloning SUMO/nanobody/ Introduction Straightforward cloning and production of biologically active proteins is central for studies in biochemistry and biophysics. During recent years, several improvements in recombinant protein expression and purification techniques have been developed. Yet many laboratories still depend on unreliable cloning techniques based on ligation of endonuclease restriction products. Especially, when cloning multiple DNA fragments into one expression vector traditional methodology requires time--consuming reiteration of the process and the cloning step frequently becomes limiting in the protein production process. Current cloning methodology relies on PCR amplification of the relevant gene using two primers designed to facilitate the subsequent cloning step. Traditionally, unique restriction sites in the flanking primer regions are digested and the PCR product is ligated into a vector containing compatible cohesive ends. Similarly, in TA--cloning a single adenosine residue added at each end of the PCR product by Taq DNA polymerase facilitates ligation into a restricted vector carrying complementary thymidine residues [1]. An alternative to these methods is ligation independent cloning (LIC). In LIC flanking sequence extensions are added to the PCR product and are by enzymatic modification converted to single stranded overhangs that are annealed to a vector carrying complementary ends [2]. Cloning of multipl...

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Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Cited by 388 publications

References 199 publications

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination

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