Expression and purification of a recombinant buforin derivative from Escherichia coli

Pyo, Sang‐Hyun; Lee, Jae-Hyun; Park, Heung-Bok; Jinsuk, Cho; Kim, Hong-Rak; Han, Byung-Hee; Park, Yeon-Sung

doi:10.1016/j.procbio.2003.07.007

Cited by 20 publications

(16 citation statements)

References 21 publications

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“…However, in the past, traditional fusion systems have given variable results of expression and have faced major problems attributed to either the inefficient cleavage of the fusion protein or cleavage within the target protein, both of which compound the difficulties of purification. Expression of the recombinant buforin IIb was achieved by Pyo et al (2004) using PurF fusion technology. Hydroxylamine was also used for chemical cleavage.…”

Section: Discussionmentioning

confidence: 99%

Expression and purification of antimicrobial peptide buforin IIb in Escherichia coli

et al. 2011

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A novel production method in Escherichia coli for an antimicrobial peptide of 21 amino acids, buforin IIb, which is a synthetic analog of buforin II, has been developed. The buforin IIb gene was cloned into the vector pET32a to construct an expression vector pET32a-buforin IIb. The fusion protein Trx-buforin IIb, purified by nickel nitrilo-triacetic acid (Ni-NTA) resin chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant buforin IIb. Purification of recombinant buforin IIb was achieved by HPLC: about 3.1 mg/l active recombinant buforin IIb with purity >99% was obtained. The recombinant buforin IIb showed antimicrobial activities that were similar to the synthetic one.

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Section: Discussionmentioning

confidence: 99%

Expression and purification of antimicrobial peptide buforin IIb in Escherichia coli

et al. 2011

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“…Many different kinds of antimicrobial peptides have been identified in recent years from various organisms, including amphibians, mammals, plants, invertebrates, and prokaryotes (3,18,22,35). To make AMPs more economically viable, researchers have sought to mass-produce AMPs using recombinant means such as insect/baculovirus (1)-, yeast (30)-, and Escherichia coli (6,12,16,19,28,29,39)-based systems. However, some of these previous studies have been limited by difficulties in recovering AMPs from engineered bacteria (especially E. coli) at high levels due to their toxicity to host cells, susceptibility to proteolytic degradation, and small size (37).…”

mentioning

confidence: 99%

“…Previous studies have investigated AMPs fused with several partners, including bovine prochymosin (11), maltose-binding protein (10,25), F4 of the E. coli intracellular protein PurF (16,28), green fluorescent protein (GFP) (36), Pap3.30 from the Pseudomonas aeruginosa bacteriophage PaP3 (29), bacterial thioredoxin (2), RepA of E. coli (39), intein (21), L-ribulokinase of Salmonella enterica serovar Typhimurium (5), the Cterminal fragment of light meromyosin (6), glutathione S-transferase (26), and immunoglobulin G (IgG)-binding domains from protein A (26). However, although some of these carrier molecules have greatly improved the stability and expression level (6,16,17,29) of the target AMP in the expression host, these strategies are still limited by low recovery yields and high levels of proteolytic degradation.…”

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confidence: 99%

Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Wei

Kim

Seo

et al. 2005

Appl Environ Microbiol

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Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein (ϳ33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP (ϳ2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.

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“…However, expressing recombinant soluble proteins, especially difficult-to-express big enzyme protein, is quite an arduous task because they often fold incorrectly and aggregate, leading to either rapid degradation or to the accumulation of inclusion bodies when expressed in E. coli. Fortunately, these problems are somewhat alleviated when the desired protein is expressed with a fusion partner, including F4 fragment of PurF (Pyo et al 2004), green fluorescent protein (GFP) (Skosyrev et al 2003) and protein PaP3.30 (Rao et al 2004). The fusion partner is often highly expressed in the host cells with an affinity tag for a particular ligand, which not only elevates the expression level of the fusion protein but also facilitates its purification (Nilsson et al 1997;Sun et al 2005).…”

Section: Discussionmentioning

confidence: 99%

Production of glutamine synthetase in Escherichia coli using SUMO fusion partner and application to l-glutamine synthesis

Wang

Cui

Yan

et al. 2011

World J Microbiol Biotechnol

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L-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine industry is expected. High efficiency of L-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v) lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS-PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel nitrilo-triacetic acid (Ni-NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni-NTA column. Finally, about 121 mg recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l L-Gln was detected by high pressure liquid chromatography (HPLC) or thinlayer chromatography. Thus, the application of SUMO technology to the expression and purification of GS potentially could be employed for the industrial production of L-Gln.

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Expression and purification of a recombinant buforin derivative from Escherichia coli

Cited by 20 publications

References 21 publications

Expression and purification of antimicrobial peptide buforin IIb in Escherichia coli

Expression and purification of antimicrobial peptide buforin IIb in Escherichia coli

Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Production of glutamine synthetase in Escherichia coli using SUMO fusion partner and application to l-glutamine synthesis

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