High-level production of a candidacidal peptide lactoferrampin in Escherichia coli by fusion expression

Yang, Yalin; Tian, Zhigang; Teng, Da; Zhang, Jun; Wang, Jiarong; Wang, Jianhua

doi:10.1016/j.jbiotec.2009.01.003

Cited by 15 publications

(8 citation statements)

References 30 publications

(49 reference statements)

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“…Of interest, TRX has also been reported to enhance the solubility of recombinant antimicrobial peptides sharing similar properties as our Kcoil peptide (e.g. positively charged and small size) (Li 2009(Li , 2011LaVallie et al 2000;Jing et al 2010;Yang et al 2009). It has been previously suggested that the good production yields recorded for cationic peptides fused to TRX were due to its anionic character (pI = 4.67) that can neutralize the high positive charge of cationic peptides, hence increasing the expression levels for this class of peptides.…”

Section: Expression Of Chimeras Corresponding To Kcoil Peptide Fused mentioning

confidence: 94%

“…It has been previously suggested that the good production yields recorded for cationic peptides fused to TRX were due to its anionic character (pI = 4.67) that can neutralize the high positive charge of cationic peptides, hence increasing the expression levels for this class of peptides. However, other studies have also suggested that the expression level might be related to (1) the mRNA stability of the partner (Yang et al 2009), (2) the chaperone-like activity of TRX (Xu et al 2007;Li 2009) or (3) its relatively low molecular-weight (~11.7 kDa), making the peptide of interest a large fraction of the fusion protein (Li 2011;Shlyapnikov et al 2008).…”

Section: Expression Of Chimeras Corresponding To Kcoil Peptide Fused mentioning

confidence: 99%

“…For this purpose, the solution mixture resulting from cleavage was directly applied to a SP-Sepharose FF column and the flow through collected. This strategy was selected as we were unable to efficiently capture the (His) 6 -tagged thioredoxin alone after TEV cleavage, even after several treatments with denaturants (e.g., high concentration of urea and guanidine hydrochloride; data not shown) most likely due to either strong electrostatic interactions between the lysine residues of the Kcoil peptide and the anionic thioredoxin (Yang et al 2009;McCoy and La Ville 2001) or the presence of a loop within the active site of thioredoxin, which may favour small peptide binding. Noteworthy, TRX protein carrying peptide of interest in this active-site loop was reported to retain its structure (LaVallie et al 1993), which may explain the inefficacity of denaturant treatment to release the Kcoil peptide.…”

Section: Optimisation Of Kcoil Peptide Purificationmentioning

confidence: 99%

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Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif

et al. 2015

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A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5 mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides.

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Section: Expression Of Chimeras Corresponding To Kcoil Peptide Fused mentioning

confidence: 94%

Section: Expression Of Chimeras Corresponding To Kcoil Peptide Fused mentioning

confidence: 99%

Section: Optimisation Of Kcoil Peptide Purificationmentioning

confidence: 99%

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Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif

et al. 2015

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“…Moreover, in fusion protein systems, enzymatic or chemical cleavage is necessary to remove fusion protein tags [20][21][22][23]. Enzymatic cleavage often causes unfavorable degradation of recombinant peptides, because widely used proteases, such as enterokinase and factor Xa, often show non-specific cleavages at unexpected sites [26,27]. Furthermore, many peptides often contain potential cleavage sites cleaved by chemicals.…”

Section: Introductionmentioning

confidence: 99%

Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization

Tomisawa

Sato

Kamiya

et al. 2015

Protein Expression and Purification

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Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed, attempts to obtain large amounts of α-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse α-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in E. coli expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies.Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of α-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step.3

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“…Luo et al (2007) have expressed recombinant bovine LF N-terminal peptide in E.coli, and the product displayed obvious antimicrobial activity. Bovine LF derivates lactoferricin (LfcinB; Feng et al 2006), LfcinB15 (Tian et al 2007) and lactoferrampin (LfampinB;Yang et al 2009) were expressed in E. coli system and displayed obvious antibacterial activity. Choi et al (2008) expressed recombinant human lactoferrin in glycoengineered Pichia pastoris to evaluate the effects of terminal Nglycan structures on immune responses.…”

Section: Introductionmentioning

confidence: 99%

Cloning, expression and characterization of Kunming mice lactoferrin and its N-lobe

Wang¹,

Tian²,

Teng³

et al. 2010

Biometals

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The lactoferrin cDNA of Kunming mice was isolated by reverse transcription polymerase chain reaction and cloned into vector pET28a(+). Its deduced amino acid sequence was analyzed and compared with lactoferrin of other species. Its secondary and tertiary structure are predicted and modeled by bioinformatics tools online. Then recombinant Kunming mice lactoferrin and its N-lobe were both expressed successfully in the Escherichia coli BL21(DE3) in the form of inclusion bodies. After purification with Ni-NTA His-Bind resin, the yield of recombinant lactoferrin was 17 mg l(-1) with purity of 92.1%, and that of lactoferrin N-lobe was 20 mg l(-1) with purity of 98.5%. The inhibition efficiency of refolded lactoferrin N-lobe against Staphylococcus aureus ATCC 25923 reaches 48.6% at the concentration of 25 micromol l(-1). However, the refolded lactoferrin (12.5 micromol l(-1)) didn't display obvious inhibition activity in the test. The expression of recombinant Kunming mice lactoferrin and its N-lobe will be helpful for the study of lactoferrin on structure, function and application in a mouse model system.

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High-level production of a candidacidal peptide lactoferrampin in Escherichia coli by fusion expression

Cited by 15 publications

References 30 publications

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif

Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization

Cloning, expression and characterization of Kunming mice lactoferrin and its N-lobe

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