[국내논문]Glutathione S-Transferase에 융합한 재조합 Hybrid Peptide Gaegurin-LL37의 대장균에서의 발현 Expression of Recombinant Hybrid Peptide Gaegurin4 and LL37 using Fusion Protein in E. coli원문보기
항균 펩타이드(Antimicrobial peptides(AMPs)는 그람 양성, 그람 음성 세균과 진균병원체에 대항하는 생명체에서 중요한 역할을 하는 물질이다. 인간의 Cathelicidin 항균 펩타이드는 임상학적으로 사용할 수 있는 여러 가지의 생물학적 활성을 가진다. 항균 펩타이드의 생산 비용은 재조합 방법으로 낮출 수가 있다. 대장균은 저렴하며 손쉬운 조작이 가능하기 때문에 다른 재조합 단백질처럼 항균 펩타이드의 발현에 훌륭한 숙주가 될 수 있다. 그러나 대장균에서의 항균 펩타이드의 과발현은 항균 펩타이드가 과발현 되었을 때 대장균에 독성을 보일 수 있으므로 어려움이 보고가 되어있다. 본 연구에서는 이러한 문제점을 극복하고자 항균 펩타이드를 Glutathione S-transferase(GST) 결합 단백질에 융합하여 항균펩타이드의 독성을 감소시키도록 설계하여 발현을 시도하였다. 이 때 발현한 항균 펩티드는 LL37과, gaegurin4과 LL37의 잡종 펩타이드 GGN4-LL37(GL32로 명명)를 GST에 융합되도록 벡터를 구축하고 설계하여 대장균에서 GST 융합단백질로 발현시켰다. 융합 단백질은 친화력 컬럼을 사용하여 분리하고 GST를 절단하여 항균펩타이드 만을 분리하였고 분리한 펩타이드는 웨스턴 블롯팅으로 확인하였고 그람 양성, 그람 음성 세균에 대하여 항균 활성을 나타내는 것을 확인하였다.
항균 펩타이드(Antimicrobial peptides(AMPs)는 그람 양성, 그람 음성 세균과 진균병원체에 대항하는 생명체에서 중요한 역할을 하는 물질이다. 인간의 Cathelicidin 항균 펩타이드는 임상학적으로 사용할 수 있는 여러 가지의 생물학적 활성을 가진다. 항균 펩타이드의 생산 비용은 재조합 방법으로 낮출 수가 있다. 대장균은 저렴하며 손쉬운 조작이 가능하기 때문에 다른 재조합 단백질처럼 항균 펩타이드의 발현에 훌륭한 숙주가 될 수 있다. 그러나 대장균에서의 항균 펩타이드의 과발현은 항균 펩타이드가 과발현 되었을 때 대장균에 독성을 보일 수 있으므로 어려움이 보고가 되어있다. 본 연구에서는 이러한 문제점을 극복하고자 항균 펩타이드를 Glutathione S-transferase(GST) 결합 단백질에 융합하여 항균펩타이드의 독성을 감소시키도록 설계하여 발현을 시도하였다. 이 때 발현한 항균 펩티드는 LL37과, gaegurin4과 LL37의 잡종 펩타이드 GGN4-LL37(GL32로 명명)를 GST에 융합되도록 벡터를 구축하고 설계하여 대장균에서 GST 융합단백질로 발현시켰다. 융합 단백질은 친화력 컬럼을 사용하여 분리하고 GST를 절단하여 항균펩타이드 만을 분리하였고 분리한 펩타이드는 웨스턴 블롯팅으로 확인하였고 그람 양성, 그람 음성 세균에 대하여 항균 활성을 나타내는 것을 확인하였다.
Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in ...
Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in large quantities, and chemical synthesis is also economically impractical, especially for long peptides. Therefore, as an alternative, heterologous expression of AMPs by recombinant techniques has been studied as a means to reduce production costs. E. coli is an excellent host for the expression of AMPs, as well as other recombinant proteins, because of the low cost involved and its easy manipulation. However, overexpression of AMPs in E. coli has been shown to cause difficulties resulting from the toxicity of the subsequently produced AMPs. Therefore, fusion expression was theorized to be a solution to this problem. In this study, AMPs were expressed as fused proteins with the glutathione S-transferase (GST) binding protein to protect against the toxicity of AMPs when expressed in E. coli. The LL37, and hybrid gaegurin and LL37 (GGN4(1-16)-LL37(17-32), which we designated as GL32, peptides were expressed as GST-fusion proteins in E. coli and the fusion proteins were then purified by affinity columns. The purified peptides were obtained by removal of GST and were confirmed by western blot analysis. The purified antimicrobial peptides then demonstrated antimicrobial activities against Gram-negative and Gram-positive bacterial strains.
Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in large quantities, and chemical synthesis is also economically impractical, especially for long peptides. Therefore, as an alternative, heterologous expression of AMPs by recombinant techniques has been studied as a means to reduce production costs. E. coli is an excellent host for the expression of AMPs, as well as other recombinant proteins, because of the low cost involved and its easy manipulation. However, overexpression of AMPs in E. coli has been shown to cause difficulties resulting from the toxicity of the subsequently produced AMPs. Therefore, fusion expression was theorized to be a solution to this problem. In this study, AMPs were expressed as fused proteins with the glutathione S-transferase (GST) binding protein to protect against the toxicity of AMPs when expressed in E. coli. The LL37, and hybrid gaegurin and LL37 (GGN4(1-16)-LL37(17-32), which we designated as GL32, peptides were expressed as GST-fusion proteins in E. coli and the fusion proteins were then purified by affinity columns. The purified peptides were obtained by removal of GST and were confirmed by western blot analysis. The purified antimicrobial peptides then demonstrated antimicrobial activities against Gram-negative and Gram-positive bacterial strains.
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제안 방법
We used the pGEX-5X3-GST vector system with inducible tac promoter. The expression vector were designed to express human AMP gene fused downstream GST gene by inserting the AMP genes into the multiple cloning sites of pGEX-5X3 having IPTG-inducible tac promoter. Between GST and AMP genes, there is Factor Xa site-specific recognition sequences, for the release of intact AMPs (Fig.
However, heterologous expression for the production of recombinant AMPs may be harmful to the host bacteria [8]. To overcome its problem, we decided to express peptides as fusion protein of glutathione S-transferase (GST) and AMPs and then purify peptide after cleaving fusion partner and examined the microcidal activity.
대상 데이터
The recombinant LL37 (rLL37) and GL32 (rGL32) were purified and were concentrated by Centricon (Amicon bioseparation, USA) as suggested by manufacturer. And the inhibitory effects of recombinant rLL37 and rGL32 were tested against three bacteria, E.
이론/모형
The recombinant LL37 (rLL37) and GL32 (rGL32) were purified and were concentrated by Centricon (Amicon bioseparation, USA) as suggested by manufacturer. And the inhibitory effects of recombinant rLL37 and rGL32 were tested against three bacteria, E. coli, B. subtilis and Staphylococcus aureus by Agar disk diffusion test method (Fig. 4A and 4B). Both the recombinant rLL37 and rGL32 like chemically-synthesized LL37 showed clear zone of inhibition against E.
성능/효과
2 shows the fusion proteins at the different steps of purification, for example, cell lysate, throw flow, wash and elution state. Intense bands corresponding to the molecular weights of the expected proteins were shown: an approximately 26 kDa protein for GST and an approximately 30 kDa protein for GST-AMPs. The presence of a band at 26 kDa may be caused by intracellular cleavage of the fusion protein, or by translational pausing at the junction between GST and the fusion partner.
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