Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System

Jang, Seongdong; Sung, Bong Hyun; Cho, Ju Hyun; Kim, Sun Chang

doi:10.1128/aem.02753-08

Cited by 20 publications

(16 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our method results in a yield of bio-produced and purified pexiganan of around 1.6 mg from 800 mL bacterial cell culture (final cultivation OD 600 ~ 2), which accounts for 31% recovery of the theoretical yield of pexiganan. This recovery is twice the recovery obtained from the previous methods producing antimicrobial peptides which have similar physicochemical properties (Jang et al 2009). …”

Section: Introductionmentioning

confidence: 72%

“…This new downstream processing method is superior to traditional thermal purification methods in terms of yield and purity (Dwyer et al 2014; Zhao et al 2015), and more competitive than chromatographic methods in terms of production cost as compared to the previous purification processes of producing antimicrobial pexiganan peptide (Jang et al 2009). We expect that this new method could offer a cost-effective and high-yield approach which can be easily generalized and adapted to recover and purify other antimicrobial peptides from microbial cell factories.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2018

View full text Add to dashboard Cite

Antimicrobial peptides (AMPs) have significant potential as alternatives to classical antibiotics. However, AMPs are currently prepared using processes which are often laborious, expensive and of low-yield, thus hindering their research and application. Large-scale methods for production of AMPs using a cost-effective approach is urgently required. In this study, we report a scalable, chromatography-free downstream processing method for producing an antimicrobial peptide, pexiganan, using recombinant Escherichia coli (E. coli). The four helix bundle structure of the unique carrier protein DAMP4 was used to facilitate a simple and cheap purification process based on a selective thermochemical precipitation. Highly pure fusion protein DAMP4var-pexiganan was obtained at high yield (around 24 mg per 800 mL cell culture with a final cultivation OD600 ~ 2). The purification yield of DAMP4var-pexiganan protein is increased twofold with a 72.9% of the protein recovery in this study as compared to the previous purification processes (Dwyer in Chem Eng Sci 105:12–21, 2014). The antimicrobial peptide pexiganan was released and activated from the fusion protein by a simple acid-cleavage. Isoelectric precipitation was then applied to separate the pexiganan peptide from the DAMP4var protein carrier. The final yield of pure bio-produced pexiganan was 1.6 mg from 800 mL of bacterial cell culture (final cultivation OD600 ~ 2). The minimum bactericidal concentration (MBC) test demonstrated that the bio-produced pexiganan has the same antimicrobial activity as chemically synthesized counterpart. This novel downstream process provides a new strategy for simple and probable economic production of antimicrobial peptides.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0541-3) contains supplementary material, which is available to authorized users.

show abstract

Section: Introductionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

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

et al. 2018

View full text Add to dashboard Cite

show abstract

“…It has been reported that the translation of an individual short peptide upstream of the gene utilizes the intrinsic helicase activities of ribosomes arriving at the second TIR, reducing the incidence of potentially inhibitory RNA structures and facilitating translation initiation at the second cistron [6,33]. When strong BEPs and MEPs were reused to express scFv, BEPs that performed well with EGFP also successfully expressed scFv, while two strong MEPs associated with the positive control failed to maintain their strong activities.…”

Section: Discussionmentioning

confidence: 99%

Bicistronic expression strategy for high‐level expression of recombinant proteins in Corynebacterium glutamicum

Liu

Zhao

Zhang

et al. 2017

Engineering in Life Sciences

View full text Add to dashboard Cite

Directly using the promoter associated with 5′‐untranslated region of a high‐protein‐abundance gene from the genome may cause low expression activity of an expression system. A bicistronic expression part containing the short 5′ coding sequence of the source gene and an embedded Shine–Dalgarno sequence can cause higher expression levels of the recombinant gene in a bicistronic cassette. Here, we evaluated two methods to construct expression parts and exploited genomic sequence sources to provide specific functional sequences to complete the expression system. The architecture of the bicistronic part increased the expression levels of target genes and performed more reliably than conventional expression parts with the same promoter and 5′ untranslated region. For Corynebacterium glutamicum, the strongest bicistronic part, HP‐BEP4, was obtained from a heterologous sequence source, leading to a 2.24‐fold increase in the expression level of fluorescent protein over constitutively expressed pXMJ19 or the production of more than 100 mg/L single‐chain variable fragment (scFv). It could meet the needs of overexpressing key genes in C. glutamicum.

show abstract

“…Jang et al . succeeded in producing a potent antimicrobial peptide, buforin ІІb, by coexpression of human gamma interferon (Jang et al 2009). To obtain a large amount of ABF-2, we decided to apply this method to ABF-2 and examine the effects of aggregation-prone partner proteins in detail.…”

Section: Discussionmentioning

confidence: 99%

“…In previous studies, translationally coupled two-cistron plasmids were used to coexpress target peptides and partner proteins (Saito et al 1987; Jang et al 2009). However, genetic manipulation was needed to construct the translationally coupled two-cistron expression systems.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system

Tomisawa

Hojo²,

Umetsu

et al. 2013

AMB Expr

View full text Add to dashboard Cite

Antibacterial factor 2 (ABF-2) is a 67-residue antimicrobial peptide derived from the nematode Caenorhabditis elegans. Although it has been reported that ABF-2 exerts in vitro microbicidal activity against a range of bacteria and fungi, the structure of ABF-2 has not yet been solved. To enable structural studies of ABF-2 by NMR spectroscopy, a large amount of isotopically labeled ABF-2 is essential. However, the direct expression of ABF-2 in Escherichia coli is difficult to achieve due to its instability. Therefore, we applied a coexpression method to the production of ABF-2 in order to enhance the inclusion body formation of ABF-2. The inclusion body formation of ABF-2 was vastly enhanced by coexpression of aggregation-prone proteins (partner proteins). By using this method, we succeeded in obtaining milligram quantities of active, correctly folded ABF-2. In addition, 15 N-labeled ABF-2 and a well-dispersed heteronuclear single quantum coherence (HSQC) spectrum were also obtained successfully. Moreover, the effect of the charge of the partner protein on the inclusion body formation of ABF-2 in this method was investigated by using four structurally homologous proteins. We concluded that a partner protein of opposite charge enhanced the formation of an inclusion body of the target peptide efficiently.

show abstract

Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System

Cited by 20 publications

References 29 publications

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

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

Bicistronic expression strategy for high‐level expression of recombinant proteins in Corynebacterium glutamicum

Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system

Contact Info

Product

Resources

About