Cost-effective downstream processing of recombinantly produced pexiganan peptide and its antimicrobial activity

Sun, Baode; Wibowo, David; Middelberg, Anton P. J.; Zhao, Chun‐Xia

doi:10.1186/s13568-018-0541-3

Cited by 28 publications

(31 citation statements)

References 35 publications

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“…The protein purification followed a standard protocol for DAMP4-based protein (Sun et al 2018). Briefly, the cell pellet was resuspended in 40 mL lysis buffer (25 mM Tris-HCl, 1 M NaCl pH 8), and then sonicated using Branson Sonifier 250 ultrasonicator (Branson Ultrasonics, Danbury, CT) at the power level of 60 W for four bursts of 30 s while keeping the cell extract cool.…”

Section: Non-chromatographic Purification Of Damp4-f-pexigananmentioning

confidence: 99%

“…DAMP4 is a designed hyperstable four-helix bundle protein (Middelberg and Dimitrijev-Dwyer 2011). and was selected here as a protein carrier to facilitate both high-level fusion protein production and non-chromatographic purification (Sun et al 2018;Wibowo et al 2017;. DAMP4 has the capacity to maintain high solubility and structural stability under high temperature (up to 110°C) and high salt concentrations under which most host protein precipitate (Dwyer et al 2014).…”

Section: Design Of Damp4-f-pexigananmentioning

confidence: 99%

“…We designed a fusion protein by fusing DAMP4 protein to a pexiganan peptide using a short linker (DPS) in our previous studies, denoted as "DAMP4-S-pexiganan" (Sun et al 2018;. DAMP4-S-pexiganan has been demonstrated to be produced in E. coli cells by incubation with 1 mM IPTG at 25°C, 180 rpm for 16 h. The same condition was applied to produce DAMP4-F-pexiganan and the SDS-PAGE gel after cell lysis showed that DAMP4-F-pexiganan produced as evidenced from the protein band at around 15 kDa, which is close to the protein's theoretical MW (15,188.78 Da) and which is absent in non-induced cultures (data not shown).…”

Section: Production Of Damp4-f-pexigananmentioning

confidence: 99%

“…In our previous work, we have developed a cost-effective purification procedure for DAMP4-fused proteins using a non-chromatography method (Sun et al 2018;Wibowo et al 2017). The method was adopted to purify DAMP4-F-pexiganan.…”

Section: Production Of Damp4-f-pexigananmentioning

confidence: 99%

“…In this study, the antimicrobial activity of DAMP4-S-pexiganan and DAMP4-F-pexiganan were compared to investigate whether the different linkers affect the antimicrobial performance of the fusion proteins. DAMP4-S-pexiganan and DAMP4-Fpexiganan were obtained from the same expression condition (incubation with 1 mM IPTG at 25°C, 180 rpm for 16 h) and purification procedures (the non-chromatographic method) (Sun et al 2018;Wibowo et al 2017;. The antimicrobial performance of the fusions was evaluated by MIC tests (Fig.…”

Section: Impact Of Linker On the Antimicrobial Activity Of The Fusionmentioning

confidence: 99%

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Design and production of a novel antimicrobial fusion protein in Escherichia coli

Sun

Wibowo

Sainsbury

et al. 2018

Appl Microbiol Biotechnol

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In recent years, antimicrobial peptides (AMPs) have attracted increasing attention. The microbial cells provide a simple, cost-effective platform to produce AMPs in industrial quantities. While AMP production as fusion proteins in microorganisms is commonly used, the recovery of AMPs necessitates the use of expensive proteases and extra purification steps. Here, we develop a novel fusion protein DAMP4-F-pexiganan comprising a carrier protein DAMP4 linked to the AMP, pexiganan, through a long, flexible linker. We show that this fusion protein can be purified using a non-chromatography approach and exhibits the same antimicrobial activity as the chemically synthesized pexiganan peptide without any cleavage step. Activity of the fusion protein is dependent on a long, flexible linker between the AMP and carrier domains, as well as on the expression conditions of the fusion protein, with low-temperature expression promoting better folding of the AMP domain. The production of DAMP4-F-pexiganan circumvents the time-consuming and costly steps of chromatography-based purification and enzymatic cleavages, therefore shows considerable advantages over traditional microbial production of AMPs. We expect this novel fusion protein, and the studies on the effect of linker and expression conditions on its antimicrobial activity, will broaden the rational design and production of antimicrobial products based on AMPs.

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Section: Non-chromatographic Purification Of Damp4-f-pexigananmentioning

confidence: 99%

Section: Design Of Damp4-f-pexigananmentioning

confidence: 99%

Section: Production Of Damp4-f-pexigananmentioning

confidence: 99%

Section: Production Of Damp4-f-pexigananmentioning

confidence: 99%

Section: Impact Of Linker On the Antimicrobial Activity Of The Fusionmentioning

confidence: 99%

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Design and production of a novel antimicrobial fusion protein in Escherichia coli

Sun

Wibowo

Sainsbury

et al. 2018

Appl Microbiol Biotechnol

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Recombinant expression and coexpression of oyster defensin and proline‐rich peptide in Komagataella phaffii

Büyükkiraz

Kesmen

2021

Biotech and App Biochem

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Proline-rich peptide (CgPrp) and defensin (CgDef), oyster (Crassostrea gigas)originated antimicrobial peptides (AMPs), were produced by the recombinant technique in Komagataella phaffii GS115 cells. For this purpose, the nucleotide sequences encoding the CgPrp and CgDef peptides were synthesized by the recursive PCR technique, and ligated in pPICZaA expression vector. Additionally, the expression cassettes of pPICZαA-CgDef and pPICZαA-CgPrp were combined using in vitro multimer ligation strategy to construct the coexpression vector pPICZaA-CgPrp-CgDef. The expression and coexpression vectors transformed into K. phaffii GS115 cells by electroporation. At the end of the 0.5% methanolinduced expression stage for 96 h, the recombinant peptides were purified from the culture medium. The concentrations of purified peptides were changed between 1.05 and 1.21 mg/L. The recombinant peptides successfully inhibited the growth of tested Gram-positive bacterial strains belonging to Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Listeria monocytogenes, and Bacillus cereus. The minimum inhibitory concentrations (MIC) of recombinant CgPrp, CgDef, and CgPrp-CgDef peptides against tested bacteria were in the range of 12. 50-25.00, 18.75-75.00, and 5.80-11.60 pg/μl, respectively. The results of the study proved that the recombinant CgPrp, CgDef, and CgPrp-CgDef peptides expressed in K. phaffii might have good potential for the inhibition of common Gram-positive pathogenic bacteria, including drug-resistant MRSA.

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Economic and ecological benefits of a leaky E. coli strain for downstream processing: a case study for staphylococcal protein A

Kastenhofer

Cataldo

Ebner

et al. 2021

J of Chemical Tech & Biotech

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BACKGROUND Downstream processing of soluble recombinant proteins from Escherichia coli is complicated by the need to access the intracellular product by cell disruption and to separate the target protein from impurities, particularly host cell protein, DNA, endotoxins and lipids. We previously demonstrated the ability of the E. coli X‐press strain to leak high amounts of product to the culture medium without sacrificing viability. In this case study, we assessed the economic and ecological benefit of this strain for downstream processing in direct comparison to the industrial standard E. coli BL21(DE3). Staphylococcal Protein A was used as a model protein. We performed recombinant protein production, primary recovery and capture by anion exchange chromatography at laboratory scale, and used the data obtained for estimating costs and resource consumption by economic modeling. RESULTS After primary recovery, the X‐press process resulted in a 1.5‐fold higher product purity, a 150‐fold lower DNA, 3.5‐fold lower endotoxin and 3.4‐fold lower lipid load compared to BL21(DE3). Consequently, anion exchanger binding capacity was increased 2.7‐fold and purity and concentration of the eluate also was increased. Extracellular protein production with X‐press resulted in a 25% reduction of costs and a 36% reduction of both water usage and water‐related CO2 emissions compared to intracellular production with BL21(DE3). CONCLUSIONS This case study performed with stapyhlococcal Protein A demonstrated the potential of E. coli X‐press to reduce costs for downstream processing and improve the environmental footprint by simplified primary recovery, lower impurity load and consequently higher chromatographic efficiency. © 2021 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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Cost-effective downstream processing of recombinantly produced pexiganan peptide and its antimicrobial activity

Cited by 28 publications

References 35 publications

Design and production of a novel antimicrobial fusion protein in Escherichia coli

Design and production of a novel antimicrobial fusion protein in Escherichia coli

Recombinant expression and coexpression of oyster defensin and proline‐rich peptide in Komagataella phaffii

Economic and ecological benefits of a leaky E. coli strain for downstream processing: a case study for staphylococcal protein A

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