Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli

Hayashi, Masahiro; Iwamoto, Shigehisa; Sato, Shinya; Sudo, Shigeo; Takagi, Mari; Sakai, Hiroshi; Hayakawa, Tohru

doi:10.1016/j.pep.2013.01.011

Cited by 17 publications

(6 citation statements)

References 20 publications

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“…Three recent papers illustrate different approaches 26-28 , with reported yields of purified proteins from batch fermentation of 7-20 mg/L. In two of these methods, a fusion protein was produced, and purification required two different adsorption steps as well as an intermediate cleavage step (plus refolding, in one case) in solution.…”

Section: Discussionmentioning

confidence: 99%

“…CysC was secreted into the bacterial periplasm and isolated using ion exchange chromatograpy followed by size exclusion chromatography. In an attempt to improve yield, Hayashi et al 26 used the peptide tag 4AaCter and codon optimization. Inclusion bodies containing the fusion protein were solubilized in urea, and the denatured protein was affinity purified on a metal affinity column.…”

Section: Introductionmentioning

confidence: 99%

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Expression, purification, and characterization of human cystatin C monomers and oligomers

Perlenfein

Murphy

2016

Protein Expression and Purification

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Human cystatin C (cysC) is a soluble basic protein belonging to the cysteine protease inhibitor family. CysC is a potent inhibitor of cathepsins - proteolytic enzymes that degrade intracellular and endocytosed proteins, remodel extracellular matrix, and trigger apoptosis. Inhibition is via tight reversible binding involving the N-terminus as well as two β-hairpin loops of cysC. As a significant component of cerebrospinal fluid, cysC has numerous other functions, including support of neural stem cell growth and differentiation. Several studies suggest that cysC may bind to the Alzheimer-related protein beta-amyloid (Aβ), and inhibit its aggregation and toxicity. Because of an increasing recognition of its important biological roles, there is considerable interest in methods to produce full-length recombinant human cysC. Several researchers have reported success, but with processes that require multiple purification steps. Here we report successful production of human cysC using an intein-based expression system and a simple one-column purification scheme. The recombinant protein so obtained was natively folded and active as an enzyme inhibitor. Unexpectedly, even mild concentration by ultrafiltration caused significant oligomerization. The oligomers are noncovalent and retain the native secondary structure and inhibitory activity of the monomer. The oligomers, but not the monomers, were highly effective at inhibiting aggregation of Aβ. These results demonstrate the critical importance of careful physicochemical characterization of recombinant cysC protein prior to evaluation of its biological functions.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression, purification, and characterization of human cystatin C monomers and oligomers

Perlenfein

Murphy

2016

Protein Expression and Purification

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“…For this tag, the unusual alkali treatment may be problematic for many proteins, and thus may hamper the general applicability beyond the four target proteins reported by the Sakai group [46].…”

Section: Purification Tags That Induce Active Protein Aggregates Inmentioning

confidence: 99%

Aggregating tags for column‐free protein purification

Lin

Zhao

Xing

et al. 2015

Biotechnology Journal

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Protein purification remains a central need for biotechnology. In recent years, a class of aggregating tags has emerged, which offers a quick, cost-effective and column-free alternative for producing recombinant proteins (and also peptides) with yield and purity comparable to that of the popular His-tag. These column-free tags induce the formation of aggregates (during or after expression) when fused to a target protein or peptide, and upon separation from soluble impurities, the target protein or peptide is subsequently released via a cleavage site. In this review, we categorize these tags as follows: (i) tags that induce inactive protein aggregates in vivo; (ii) tags that induce active protein aggregates in vivo; and (iii) tags that induce soluble expression in vivo, but aggregates in vitro. The respective advantages and disadvantages of these tags are discussed, and compared to the three conventional tags (His-tag, maltose-binding protein [MBP] tag, and intein-mediated purification with a chitin-binding tag [IMPACT-CN]). While this new class of aggregating tags is promising, more systematic tests are required to further the use. It is conceivable, however, that the combination of these tags and the more traditional columns may significantly reduce the costs for resins and columns, particularly for the industrial scale.

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“…It was demonstrated that fusion with the 4AaCter-tag facilitates the formation of protein inclusion bodies composed of fused heterologous protein in E. coli (Hayakawa et al 2010). Fusion with the 4AaCter-tag was shown to dramatically increase protein production and simplify product purification for a number of recombinant proteins, including syphilis antigens, TpNs (Hayakawa et al 2010), markers of renal dysfunction, cystatin C (Hayashi et al 2013), and scorpion toxin as a bioinsecticide (Matsumoto et al 2014). By contrast, Cry toxins with smaller-sized protoxins (70 and 30 kDa-types) do not contain the polypeptide corresponding to the C-terminal half of 130 kDa-type protoxin, and crystallization of these proteins is thus thought to require an accessory protein such as P20 and/or ORF2 (Agaisse and Lereclus 1995).…”

Section: Introductionmentioning

confidence: 99%

Potency of the mosquitocidal Cry46Ab toxin produced using a 4AaCter-tag, which facilitates formation of protein inclusion bodies in Escherichia coli

et al. 2017

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Cry46Ab toxin derived from Bacillus thuringiensis strain TK-E6 shows mosquitocidal activity against Culex pipiens pallens Coquillett (Diptera: Culicidae) larvae as well as preferential cytotoxicity against human cancer cells. In B. thuringiensis cells, Cry46Ab is produced and accumulates as a protein crystal that is processed into the active 29-kDa toxin upon solubilization in the alkaline environment of the insect midgut. The Cry46Ab protoxin is 30 kDa and is therefore thought to require an accessory protein such as P20 and/or ORF2 for efficient crystal formation. For efficient production of alkali-soluble inclusion bodies of recombinant Cry46Ab in Escherichia coli, the potency of the 4AaCter-tag was assessed in the present study. The 4AaCter-tag is a polypeptide derived from the C-terminal region of B. thuringiensis Cry4Aa toxin and facilitates the formation of alkali-soluble protein inclusion bodies in E. coli. Fusion with the 4AaCter-tag enhanced both Cry46Ab production and the formation of Cry46Ab inclusion bodies. In addition, upon optimization of protein expression procedures, the Cry46Ab-4AaCter inclusion bodies showed mosquitocidal activity and stability in aqueous environments comparable to Cry46Ab without the 4AaCter-tag. Our study suggests that use of the 4AaCter-tag is a straightforward approach for preparing formulations of smaller-sized Cry toxins such as Cry46Ab in E. coli.

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Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli

Cited by 17 publications

References 20 publications

Expression, purification, and characterization of human cystatin C monomers and oligomers

Expression, purification, and characterization of human cystatin C monomers and oligomers

Aggregating tags for column‐free protein purification

Potency of the mosquitocidal Cry46Ab toxin produced using a 4AaCter-tag, which facilitates formation of protein inclusion bodies in Escherichia coli

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