Expression and characterization of a housefly cecropin gene in the methylotrophic yeast, Pichia pastoris

Jin, Fengliang; Xu, Xi; Zhang, Wenqing; Gu, De-Xiang

doi:10.1016/j.pep.2006.03.008

Cited by 44 publications

(46 citation statements)

References 29 publications

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“…The cecropin protein Mdcec was successfully expressed in the methylotrophic yeast P. pastoris (21) and in E. coli (47), with 1.2 mg pure recombinant Mdcec obtained from 100 ml of crude yeast extract (21) and 11.2 mg pure active recombinant Mdmcec obtained from 1 liter of E. coli culture (47). In this study, the B. subtilis expression system for the constitutive expression of recombinant cecropin was adopted.…”

Section: Discussionmentioning

confidence: 99%

“…In order to facilitate the expression of cecropin AD, this system included the SUMO protease gene, which could be released under appropriate conditions, thus ensuring the cleavage of the target gene (4). In addition, the modified expression system required no inducer during the process of expression (21), and it produced no inclusion bodies (34,47). Therefore, the system had the advantage of a high efficiency of expression at a low cost and was easy to operate, making it applicable to industrial production.…”

Section: Discussionmentioning

confidence: 99%

“…Several recombinant cecropins have been expressed in Escherichia coli (47) and Pichia pastoris (21,48) expression systems. However, the expression of foreign proteins in E. coli often leads to the formation of densely packed denatured peptide molecules in the form of insoluble particles called inclusion bodies.…”

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confidence: 99%

“…Inclusion body proteins are devoid of biological activity, and an elaborate process involving solubilization, refolding, and purification is needed to partially recover a functionally active product (34). While use of a yeast expression system such as P. pastoris may solve the problem of posttranslational modifications (21), it requires a significant investment during the later period of batch cultivation, which renders it uneconomical.…”

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confidence: 99%

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Novel Expression Vector for Secretion of Cecropin AD in Bacillus subtilis with Enhanced Antimicrobial Activity

Chen

Zhu

Cao

et al. 2009

Antimicrob Agents Chemother

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Cecropin AD, a chimeric antimicrobial peptide obtained from cecropins, is effective at killing specific microorganisms. However, a highly efficient expression system is still needed to allow for commercial application of cecropin AD. For the exogenous expression of cecropin AD, we fused the cecropin AD gene with a small ubiquitin-like modifier (SUMO) gene and a signal peptide of SacB, while a Bacillus subtilis expression system was constructed based on Bacillus subtilis cells genetically modified by the introduction of an operon including an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible Spac promoter, a signal peptide of amyQ, and the SUMO protease gene. The recombinant cecropin AD was expressed, and 30.6 mg of pure recombinant protein was purified from 1 liter of culture supernatant. The purified cecropin AD displayed antimicrobial activity against some pathogens, such as Staphylococcus aureus and Escherichia coli, and was especially effective toward Staphylococcus aureus, with MICs of <0.05 M (0.2 g/ml). Stability analysis results showed that the activity of cecropin AD was not influenced by temperatures as high as 55°C for 20 min; however, temperatures above 85°C (for 20 min) decreased the antimicrobial activity of cecropin AD. Varying the pH from 4.0 to 9.0 did not appear to affect the activity of cecropin AD, but some loss of potency was observed at pH values lower than pH 4.0. Under the challenge of several proteases (proteinase K, trypsin, and pepsin), cecropin AD maintained functional activity. The results indicated that the recombinant product expressed by the designed Bacillus subtilis expression system was a potent antimicrobial agent and could be applied to control infectious diseases of farm animals or even humans.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Novel Expression Vector for Secretion of Cecropin AD in Bacillus subtilis with Enhanced Antimicrobial Activity

Chen

Zhu

Cao

et al. 2009

Antimicrob Agents Chemother

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“…Following centrifugation at 1,000 x g for 5 min, the pellet was re-suspended to achieve an OD 600 of 1.0 in 300 ml BMMYH (BMGYH with 1% glycerol replaced with 0.8% methanol) to induce expression. Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).…”

Section: Construction Of Recombinant Expression Plasmidsmentioning

confidence: 99%

Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration

Song

Ding

Jin

et al. 2016

Molecular Medicine Reports

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Abstract. Fibroblast growth factor (FGF)21 functions in the maintenance of glucose homeostasis and exerts protective effects on the liver, heat and kidneys. However, the roles of FGF21 in other tissue types are yet to be fully elucidated. The present study detected elevated expression levels of FGF21 in skin tissue. Furthermore, it was revealed that FGF21 expression in the skin was induced upon wounding. In addition, β-klotho expression was detected in the skin tissue. To examine the role of FGF21 in the wound healing process, recombinant human (h)FGF21 was expressed in a the yeast strain Pichia (P.) pastoris, a well-known system for recombinant protein production. Based on the sequence of hFGF21 and the optimal codon of P. pastoris, codon-optimized FGF21 open reading frame sequences were obtained using seven pairs of 55-59-nt primers with seven rounds of PCR. The recombinant FGF21 was purified and its function was examined in human fibroblast cells using a wound healing cell migration assay. Treatment with FGF21 promoted cell migration, which is an important step in wound healing. Furthermore, FGF21 treatment enhanced the activity of c-Jun N-terminal kinase, a key regulator in fibroblast-cell migration. In conclusion, FGF21 is induced after wounding and FGF21 expressed and purified from yeast markedly accelerates wound healing. The present study was the first to elucidate the function of FGF21 in skin tissues and provided a theoretical basis for the use of FGF21 in the treatment of skin wounds. IntroductionThe mammalian genome contains 23 fibroblast growth factors (FGFs) (1), which have essential roles in metabolism and development. FGFs have been identified to be involved in processes of embryogenesis, including gastrulation, somitogenesis, body plan formation and organogenesis, as well as in skin wound healing (2-7). Initially, 1, a member of the FGF family, was reported to be preferentially expressed in the liver (8). However, recent studies have identified that FGF21 is inducible by starvation or certain drugs, and have reported on its diverse functions in glucose homeostasis as well as hepatoand cardioprotection (9-11). FGF19, FGF21 and FGF23 belong to the FGF19 sub-family. Among them, FGF21 primarily activates FGF receptor (FGFR)1c, for which co-receptor β-klotho is required (12,13).Previous efforts to produce recombinant human FGF21 (rhFGF21) using an Escherichia (E.) coli system resulted in low expression of soluble protein, indicating that the majority of recombinant protein localized in inclusion bodies. Although the small ubiquitin-like modifier (SUMO) fusion system has been shown to facilitate the soluble expression and enhance the production of bioactive rhFGF21 (14), additional steps are required for the removal of tags from the protein expressed in vitro. At present, Pichia (P.) pastoris (yeast) is among the most successful eukaryotic protein expression systems. Compared with mammalian cells, culture of P. pastoris requires simple and cheap growth media and conditions, and P. pastoris is known to...

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Considerations for the process development of insect‐derived antimicrobial peptide production

Müller

Salzig

Czermak

2014

Biotechnology Progress

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Antimicrobial peptides (AMPs) could evolve into new therapeutic lead molecules against multi-resistant bacteria. As insects are a rich source of AMP, the identification and characterization of insect-derived AMPs is particularly emphasized. One challenge of bringing these molecules into market, e.g., as a drug, is to develop a cost-efficient large-scale production process. Due to the fact that a direct AMP isolation from insects is not economical and that chemical synthesis is recommended for peptide sizes below 40 amino acids, a viable option is heterologous AMP production. Therefore, previous knowledge concerning the expression of larger proteins can be adapted, but due to the AMP nature (e.g., small size, bactericide) additional challenges have to be faced during up and downstream processing. Nonetheless the bottleneck for large-scale AMP production is the same as for proteins; mainly the downstream process. This review introduces opportunities for insect-derived AMP production, like the choice of the expression system (based on previously derived data), depending on the AMP nature, as well as new purification strategies like elastin-like peptide/intein based purification strategies. All of these aspects are discussed with regard to large-scale processes and costs.

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Expression and characterization of a housefly cecropin gene in the methylotrophic yeast, Pichia pastoris

Cited by 44 publications

References 29 publications

Novel Expression Vector for Secretion of Cecropin AD in Bacillus subtilis with Enhanced Antimicrobial Activity

Novel Expression Vector for Secretion of Cecropin AD in Bacillus subtilis with Enhanced Antimicrobial Activity

Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration

Considerations for the process development of insect‐derived antimicrobial peptide production

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