KR101622373B1 - Method for producing antimicrobial peptide using insoluble green fluorescent protein as a scaffold - Google Patents
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- KR101622373B1 KR101622373B1 KR1020140183350A KR20140183350A KR101622373B1 KR 101622373 B1 KR101622373 B1 KR 101622373B1 KR 1020140183350 A KR1020140183350 A KR 1020140183350A KR 20140183350 A KR20140183350 A KR 20140183350A KR 101622373 B1 KR101622373 B1 KR 101622373B1
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- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C12N15/62—DNA sequences coding for fusion proteins
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
Description
본 발명은 녹색형광단백질(Green Fluorescent protein, GFP)과 항균 펩타이드(Antimicrobial peptide, AMP)가 결합된 융합단백질 및 이를 이용한 항균 펩타이드의 대량 제조방법에 관한 것으로서, 더욱 상세하게는 항균 펩타이드를 대장균 내에서 불용성으로 발현되는 녹색형광단백질과 융합시켜 대장균에 도입 및 발현시킨 후 녹색형광단백질을 제거하고 항균 펩타이드를 수득하는 과정을 통하여 항균 펩타이드를 대량으로 제조하는 방법에 관한 것이다. The present invention relates to a fusion protein comprising a green fluorescent protein (GFP) and an antimicrobial peptide (AMP), and a method for mass-producing an antimicrobial peptide using the fusion protein. More particularly, The present invention relates to a method for producing a large amount of an antimicrobial peptide through a process of introducing and expressing an insoluble green fluorescent protein into E. coli and then removing a green fluorescent protein and obtaining an antimicrobial peptide.
미생물을 비롯하여 고등생물에 이르기까지 모든 생물은 외부의 유해환경으로부터 자신을 보호하기 위해 자기 자신만의 고유 방어체계를 갖추고 있다. 주변에 존재하는 미생물과 심지어 균사류에 이르기까지 항균활성을 보이는 자기 방어 물질인 항균 펩타이드를 생산하여 선천적 면역체계를 갖춤으로써, 항원-항체 반응 이전에 일차적으로 외부 유해요소로부터 자기 자신을 보호하는 것으로 알려져 있다.Every organism, from microorganisms to higher organisms, has its own unique defense system to protect itself from external harmful environments. By producing an antimicrobial peptide that is a self-defense substance that shows antimicrobial activity to the surrounding microorganisms and even mycelium, it has a innate immune system, which protects itself from external harmful factors before the antigen-antibody reaction It is known.
한편, 인류는 페니실린 이후 수많은 종류의 항생제가 개발되어 사용되어 왔으나, 근래에 들어서 이들 항생제에 내성을 가지는 균주들이 과거와는 달리 빠른 속도로 등장하고 있다. 특히 서로 다른 두 개 이상의 항생, 항균제에 내성을 나타내는 다중약물내성 미생물이 급증하고 있어, 이들 내성 균주를 퇴치할 수 있는 새로운 작용 메커니즘을 가지는 항생제의 개발이 시급하다. On the other hand, since penicillin, many kinds of antibiotics have been developed and used in humankind, but recently, strains resistant to these antibiotics have emerged at a rapid rate. Especially, multi-drug resistance microorganisms showing resistance to two or more different antibiotics and antimicrobial agents are soaring, and it is urgent to develop antibiotics having a new action mechanism capable of eliminating these resistant strains.
이로 인해 천연에 존재하는 항균 펩타이드는 새로운 항생제의 후보물질로서 대두되었고, 이는 기존에 사용되고 있는 화합물성 항생제와 다른 작용 기작을 통하여 항균 활성을 나타내므로 항생제 내성 균주에 대한 문제를 해결할 수 있을 것으로 기대되고 있다. As a result, antimicrobial peptides existing in nature have emerged as candidates for new antibiotics, and they are anticipated to solve the problem of antibiotic resistant strains because they exhibit antimicrobial activity through a mechanism of action other than the existing compound antibiotics have.
이에 따라 자연계에 존재하는 항균 펩타이드들을 그대로 이용하거나 유사체들을 합성하여 이용하고자 하는 시도들이 있었으나, 항균 펩타이드는 항균 활성의 증가와 동시에 세포독성의 척도인 적혈구 용혈현상도 동시에 증가시키기 때문에 실제적인 응용에 많은 제약을 받고 있다. Therefore, there have been attempts to utilize the antimicrobial peptides existing in nature or to synthesize analogues. However, since the antimicrobial peptides simultaneously increase the antifungal activity and the red blood cell hemolysis, which is a measure of cytotoxicity, It is constrained.
또한, 항균 펩타이드의 상용화란 측면에서 가장 중요한 요소인 대량생산을 위한 연구도 다양하게 시도되어 왔으나 현재까지 산업적으로 활용할만한 수준에 이르지 못하였다. 왜냐하면 화학합성으로 항균 펩타이드를 생산하는 경우에는 경제성이 낮고, 미생물을 이용한 유전공학적 기법으로 항균 펩타이드를 생산하는 경우에는 경제성은 있으나, 발현된 항균 펩타이드가 숙주 미생물의 성장을 저해하여 펩타이드 생산의 수율이 매우 낮다는 문제점이 있기 때문이다. In addition, various studies for mass production, which is the most important factor in the commercialization of antimicrobial peptides, have been attempted, but they have not reached the level that can be industrially utilized until now. In the case of producing an antimicrobial peptide by chemical synthesis, it is economically disadvantageous. In the case of producing an antimicrobial peptide by a genetic engineering technique using microorganisms, it is economical. However, since the expressed antimicrobial peptide inhibits the growth of the host microorganism, It is very low.
이에 따라, 본 발명자는 유전자 조작이 용이하고 경제적인 대장균 발현 시스템을 이용하되, 대장균 내에서 불용성으로 발현되는 녹색형광단백질을 지지체로서 항균 펩타이드에 결합시킨 융합단백질의 발현을 유도한 후 상기 융합단백질로부터 항균 펩타이드만을 분리하는 과정을 통하여 펩타이드를 제조함으로써, 단백질 가수분해로 인한 항균 펩타이드 생성 저해 및 항균 펩타이드가 대장균의 성장을 저해하여 펩타이드의 생산 수율이 낮아지는 문제를 해결함으로써, 간단하고 경제적으로 항균 펩타이드를 대량 제조하는 방법에 관한 발명을 완성하였다. Accordingly, the present inventors have succeeded in inducing expression of a fusion protein in which a green fluorescent protein, which is insoluble in E. coli, is bound to an antimicrobial peptide as a supporter, using an E. coli expression system that is easy and economical in gene manipulation, By solving the problem of inhibition of the production of antimicrobial peptides due to protein hydrolysis and the problem that the antimicrobial peptide inhibits the growth of E. coli and the production yield of the peptide is lowered by producing only the antimicrobial peptide alone, the antimicrobial peptide The present invention has been completed.
관련 종래기술로는 대한민국 등록특허 제10-0958095호(번역 동반 시스템을 이용한 항균 펩타이드의 대량 발현방법), 대한민국 공개특허 제10-2012-0062504호(세포표면에서 발현되는 항균 펩타이드 다중합체) 등이 있다. Korean Patent No. 10-0958095 (a method for mass-expression of an antimicrobial peptide using a translational entrainment system) and Korean Patent Laid-Open No. 10-2012-0062504 (an antimicrobial peptide-type polysaccharide expressed on a cell surface) have.
본 발명의 목적은, 지지체로서 불용성의 녹색형광단백질을 이용하고 또한 대장균 발현 시스템을 활용하여 항균 펩타이드를 제조함으로써, 제조공정이 간단하고 경제적이며 생산 수율이 높은 항균 펩타이드의 제조방법을 제공함에 있다. An object of the present invention is to provide a method for producing an antimicrobial peptide which is simple and economical in production process and yielded high yield by using an insoluble green fluorescent protein as a support and an antimicrobial peptide using an E. coli expression system.
상기 목적을 달성하기 위하여, 본 발명은 녹색형광단백질(Green Fluorescent protein, GFP)과 항균 펩타이드(Antimicrobial peptide, AMP)가 결합된 융합단백질을 제공한다. In order to achieve the above object, the present invention provides a fusion protein in which a green fluorescent protein (GFP) and an antimicrobial peptide (AMP) are combined.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PG1이 결합된 융합단백질을 코딩하는 유전자를 제공한다. The present invention also provides a gene coding for a fusion protein in which the green fluorescent protein and the antimicrobial peptide PG1 are bound.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PMAP36이 결합된 융합단백질을 코딩하는 유전자를 제공한다. In addition, the present invention provides a gene encoding the fusion protein in which the green fluorescent protein and the antibacterial peptide PMAP36 are combined.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PG1이 결합된 융합단백질을 코딩하는 유전자를 포함하는 발현벡터를 제공한다.The present invention also provides an expression vector comprising the gene encoding the fusion protein in which the green fluorescent protein and the antimicrobial peptide PG1 are combined.
또한, 본 발명은 녹색형광단백질과 항균 펩타이드 PMAP36이 결합된 융합단백질을 코딩하는 유전자를 포함하는 발현벡터를 제공한다. In addition, the present invention provides an expression vector comprising a gene encoding a fusion protein in which a green fluorescent protein and an antibacterial peptide PMAP36 are combined.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PG1이 결합된 융합단백질을 코딩하는 유전자를 포함하는 발현벡터로 형질전환된 대장균 형질전환체를 제공한다. The present invention also provides an E. coli transformant transformed with an expression vector comprising a gene encoding the fusion protein in which the green fluorescent protein and the antimicrobial peptide PG1 are combined.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PMAP36이 결합된 융합단백질을 코딩하는 유전자를 포함하는 발현벡터로 형질전환된 대장균 형질전환체를 제공한다. The present invention also provides an E. coli transformant transformed with an expression vector comprising the gene encoding the fusion protein in which the green fluorescent protein and the antibacterial peptide PMAP36 are combined.
또한, 본 발명은 상기 항균 펩타이드 제조를 위한 녹색형광단백질의 아미노산 서열 중 1번에서 172번 사이의 DNA 증폭을 위한 서열번호 3(정방향 프라이머) 및 서열번호 4(역방향 프라이머)로 이루어진 중합효소 연쇄반응용 프라이머 세트를 제공한다. The present invention also relates to a method for amplifying DNA fragments comprising the steps of: (1) amplifying DNA fragments from 1 to 172 amino acids of a green fluorescent protein for producing the above antimicrobial peptide, (3) a forward primer, and (4) Lt; / RTI >
또한, 본 발명은 상기 항균 펩타이드 제조를 위한 녹색형광단백질의 아미노산 서열 중 173번에서 3′말단 사이의 DNA 증폭을 위한 서열번호 5(정방향 프라이머) 및 서열번호 6(역방향 프라이머) 으로 이루어진 중합효소 연쇄반응용 프라이머 세트를 제공한다. The present invention also relates to a method for amplifying DNA from the 173rd to 3'end of the amino acid sequence of the green fluorescent protein for the production of the above antimicrobial peptide using the sequence of SEQ ID NO: 5 (forward primer) and SEQ ID NO: 6 (reverse primer) A primer set for reaction is provided.
또한, 본 발명은 (1) 녹색형광단백질을 코딩하는 유전자 및 항균 펩타이드를 코딩하는 유전자를 재조합 하는 단계; (2) 상기 재조합된 유전자를 클로닝(cloning)하여 대장균 형질전환체를 제조하는 단계; (3) 상기 제조된 대장균 형질전환체를 배양하여 재조합 단백질의 발현을 유도하는 단계; (4) 상기 발현된 재조합 단백질을 분리 후 정제하는 단계; 및 (5) 상기 정제된 재조합 단백질에서 항균 펩타이드를 분리 후 정제하는 단계; 를 포함하는 항균 펩타이드의 제조방법을 제공한다. (1) recombining a gene encoding a green fluorescent protein and a gene encoding an antimicrobial peptide; (2) cloning the recombinant gene to prepare an E. coli transformant; (3) culturing the E. coli transformant to induce expression of the recombinant protein; (4) separating and purifying the expressed recombinant protein; And (5) separating and purifying the antimicrobial peptide from the purified recombinant protein; And a method for producing the antimicrobial peptide.
상기 (1)단계에서 항균 펩타이드는 서열번호 1의 염기서열로 구성된 프로테그린1(Protegrin1, PG1) 또는 서열번호 2의 염기서열로 구성된 돼지 골수 유래 항균 펩타이드36(Pig myeloid antibacterial peptide36, PMAP36)인 것을 특징으로 한다. In step (1), the antimicrobial peptide comprises Protegrin 1 (PG1) consisting of the nucleotide sequence of SEQ ID NO: 1 or Pig myeloid antibacterial peptide 36 (PMAP36) consisting of the nucleotide sequence of SEQ ID NO: 2 .
상기와 같은 본 발명에 따르면, 간단하고 경제적이며 생산 수율이 높은 항균 펩타이드의 대량 제조방법을 제공함으로써, 다중약물내성 미생물의 급증으로 인하여 새로운 항생제의 개발이 시급한 현 시점에서, 천연에 존재하는 항균 펩타이드를 제약 및 사료 산업 등에 이용할 수 있는 효과가 있다. The present invention provides a method for mass production of antimicrobial peptides that is simple, economical, and has a high production yield. Thus, it is necessary to develop new antibiotics due to the proliferation of multiple drug resistant microorganisms. Can be used in pharmaceutical and feed industries.
도 1은 지지체로서 불용성 녹색형광단백질과 항균 펩타이드의 융합 및 대장균 발현시스템을 이용한 항균 펩타이드 제조과정의 모식도이다.
도 2는 r5M-172PG1173 유전자가 삽입된 pET30b 발현벡터가 도입된 대장균 내에서 단백질의 발현이 유도된 것과 유도되지 않은 것의 대장균 성장곡선을 광학 밀도(optical density)를 통하여 측정한 그래프이다.
도 3는 대장균 내에서 r5M-172PG1173 유전자가 삽입된 pET30b 발현벡터의 시간 경과(3h, 4h, 5h)에 따른 r5M-172PG1173 단백질 생산을 전체 세포 단백질을 샘플로 하여 SDS-PAGE로 확인한 것이다.
도 4a는 발현된 r5M-172PG1173 단백질을 각 단계마다 SDS-PAGE를 통하여 확인한 것이다.
도 4b는 r5M-172PMAP36173 단백질을 각 단계마다 SDS-PAGE를 통하여 확인한 것이다.
도 5a 및 도 5b는 tris-tricine SDS-PAGE 로 단백질 정제의 각 단계 마다 추출되는 단백질 샘플의 순도를 분석한 것이다.
도 5c는 정제된 PG1을 western blot을 통하여 확인한 것이다. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a process for producing an antimicrobial peptide using fusion of an insoluble green fluorescent protein and an antimicrobial peptide as a support and an Escherichia coli expression system. FIG.
FIG. 2 is a graph showing the growth curve of E. coli in which protein expression was induced and not induced in E. coli into which pET30b expression vector with r5M-172PG1173 gene was introduced, through optical density.
FIG. 3 shows the r5M-172PG1173 protein production by SDS-PAGE using the whole cell protein as a sample according to the lapse of time (3h, 4h, 5h) of pET30b expression vector into which r5M-172PG1173 gene was inserted in E. coli.
Figure 4a shows the expression of the expressed r5M-172PG1173 protein by SDS-PAGE at each step.
FIG. 4B shows the r5M-172PMAP36173 protein obtained by SDS-PAGE at each step.
Figures 5A and 5B are purity analyzes of protein samples extracted at each step of protein purification by tris-tricine SDS-PAGE.
FIG. 5C shows the purified PG1 through western blotting.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
상기 목적을 달성하기 위하여, 본 발명은 녹색형광단백질(Green Fluorescent protein, GFP)과 항균 펩타이드(Antimicrobial peptide, AMP)가 결합된 융합단백질을 제공한다. In order to achieve the above object, the present invention provides a fusion protein in which a green fluorescent protein (GFP) and an antimicrobial peptide (AMP) are combined.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PG1이 결합된 융합단백질을 코딩하는 유전자를 제공한다. The present invention also provides a gene coding for a fusion protein in which the green fluorescent protein and the antimicrobial peptide PG1 are bound.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PMAP36이 결합된 융합단백질을 코딩하는 유전자를 제공한다. In addition, the present invention provides a gene encoding the fusion protein in which the green fluorescent protein and the antibacterial peptide PMAP36 are combined.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PG1이 결합된 융합단백질을 코딩하는 유전자를 포함하는 발현벡터를 제공한다.The present invention also provides an expression vector comprising the gene encoding the fusion protein in which the green fluorescent protein and the antimicrobial peptide PG1 are combined.
또한, 본 발명은 녹색형광단백질과 항균 펩타이드 PMAP36이 결합된 융합단백질을 코딩하는 유전자를 포함하는 발현벡터를 제공한다. In addition, the present invention provides an expression vector comprising a gene encoding a fusion protein in which a green fluorescent protein and an antibacterial peptide PMAP36 are combined.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PG1이 결합된 융합단백질 을 코딩하는 유전자를 포함하는 발현벡터로 형질전환된 대장균 형질전환체를 제공한다. The present invention also provides an E. coli transformant transformed with an expression vector comprising a gene encoding the fusion protein in which the green fluorescent protein and the antimicrobial peptide PG1 are combined.
또한, 본 발명은 상기 녹색형광단백질과 항균 펩타이드 PMAP36이 결합된 융합단백질을 코딩하는 유전자를 포함하는 발현벡터로 형질전환된 대장균 형질전환체를 제공한다. The present invention also provides an E. coli transformant transformed with an expression vector comprising the gene encoding the fusion protein in which the green fluorescent protein and the antibacterial peptide PMAP36 are combined.
또한, 본 발명은 상기 항균 펩타이드 제조를 위한 녹색형광단백질의 아미노산 서열 중 1번에서 172번 사이의 DNA 증폭을 위한 서열번호 3(정방향 프라이머) 및 서열번호 4(역방향 프라이머)로 이루어진 중합효소 연쇄반응용 프라이머 세트를 제공한다. The present invention also relates to a method for amplifying DNA fragments comprising the steps of: (1) amplifying DNA fragments from 1 to 172 amino acids of a green fluorescent protein for producing the above antimicrobial peptide, (3) a forward primer, and (4) Lt; / RTI >
또한, 본 발명은 상기 항균 펩타이드 제조를 위한 녹색형광단백질의 아미노산 서열 중 173번에서 3′말단 사이의 DNA 증폭을 위한 서열번호 5(정방향 프라이머) 및 서열번호 6(역방향 프라이머)으로 이루어진 중합효소 연쇄반응용 프라이머 세트를 제공한다. The present invention also relates to a method for amplifying DNA from the 173rd to 3'end of the amino acid sequence of the green fluorescent protein for the production of the above antimicrobial peptide using the sequence of SEQ ID NO: 5 (forward primer) and SEQ ID NO: 6 (reverse primer) A primer set for reaction is provided.
또한, 본 발명은 (1) 녹색형광단백질을 코딩하는 유전자 및 항균 펩타이드를 코딩하는 유전자를 재조합 하는 단계; (2) 상기 재조합된 유전자를 클로닝(cloning)하여 대장균 형질전환체를 제조하는 단계; (3) 상기 제조된 대장균 형질전환체를 배양하여 재조합 단백질의 발현을 유도하는 단계; (4) 상기 발현된 재조합 단백질을 분리 후 정제하는 단계; 및 (5) 상기 정제된 재조합 단백질에서 항균 펩타이드를 분리 후 정제하는 단계; 를 포함하는 항균 펩타이드의 제조방법을 제공한다. (1) recombining a gene encoding a green fluorescent protein and a gene encoding an antimicrobial peptide; (2) cloning the recombinant gene to prepare an E. coli transformant; (3) culturing the E. coli transformant to induce expression of the recombinant protein; (4) separating and purifying the expressed recombinant protein; And (5) separating and purifying the antimicrobial peptide from the purified recombinant protein; And a method for producing the antimicrobial peptide.
상기 (1)단계에서 항균 펩타이드는 서열번호 1의 염기서열로 구성된 프로테그린1(Protegrin1, PG1) 또는 서열번호 2의 염기서열로 구성된 돼지 골수 유래 항균 펩타이드36(Pig myeloid antibacterial peptide36, PMAP36)인 것을 특징으로 한다. In step (1), the antimicrobial peptide comprises Protegrin 1 (PG1) consisting of the nucleotide sequence of SEQ ID NO: 1 or Pig myeloid antibacterial peptide 36 (PMAP36) consisting of the nucleotide sequence of SEQ ID NO: 2 .
본 발명에서는 대장균 발현 시스템을 이용하여 항균 펩타이드를 대량으로 제조하는 과정에 있어서, 대장균 내에서 단백질 가수분해효소에 의한 단백질 분해 및 항균 펩타이드가 대장균의 성장을 저해하여 펩타이드의 생산 수율이 낮아지는 문제를 해결하기 위하여 항균 펩타이드의 지지체로서 녹색형광단백질을 이용하였다. 즉, 대장균 내에서 녹색형광단백질과 항균 펩타이드가 결합된 불용성의 융합단백질의 구조로 발현을 유도함으로써 항균 펩타이드 손상을 막고, 항균 펩타이드의 발현이 숙주에 미치는 성장저해 효과를 극소화하였다. In the present invention, in the process of producing a large amount of antimicrobial peptides using an E. coli expression system, proteolysis by proteolytic enzymes in E. coli and antimicrobial peptides inhibit the growth of E. coli, thereby lowering the yield of peptide production To solve this problem, a green fluorescent protein was used as a supporter of an antimicrobial peptide. That is, by inducing expression in the structure of an insoluble fusion protein in which the green fluorescent protein and the antimicrobial peptide are bound in E. coli, the antimicrobial peptide damage is prevented and the growth inhibition effect of the antimicrobial peptide on the host is minimized.
한편, 다양한 생물체로부터 지금까지 발견된 항균 펩타이드는 구조적으로 세 개의 그룹으로 나뉘어진다. 첫 번째는, 시스테인이 풍부한 β-쉬트(sheet) 펩타이드 이고, 두 번째는 α-회전형(helical)의 양친화성 분자이며, 세 번째는 프롤린이 풍부한 펩타이드이다. 이들 항균 펩타이드의 다양한 구조는 펩타이드 아미노산 서열에 의해 결정되며, 이러한 구조는 펩타이드의 항균 활성과 밀접한 관계를 가지고 있는 것으로 알려져 있다. On the other hand, the antimicrobial peptides discovered so far from various organisms are structurally divided into three groups. The first is the cysteine-rich β-sheet peptide, the second is the α-helical amphipathic molecule, and the third is the proline-rich peptide. The various structures of these antimicrobial peptides are determined by peptide amino acid sequences, and these structures are known to have a close relationship with the antimicrobial activity of the peptides.
본 발명에서는 두 개의 이황화 결합에 의해 안정화 된, 전형적인 역-평행 베타-헤어핀 구조를 가지는 프로테그린1(Protegrin1, PG1)을 사용하였는데, 이는 돼지 백혈구에서 최초로 발견되었다. 이외에도 나선(helix) 구조를 가지는 돼지 골수 유래 항균 펩타이드36(Pig myeloid antibacterial peptide36, PMAP36)을 사용하였으나 이에 한정되지 아니한다. In the present invention, Protegrin1 (PG1), which is stabilized by two disulfide bonds and has a typical reverse-parallel beta-hairpin structure, was first found in porcine white blood cells. In addition, Pig myeloid antibacterial peptide 36 (PMAP36), which has a helix structure, is used but not limited thereto.
본 발명에서 제조된 항균 펩타이드의 제조방법을 상세히 살펴보면, 먼저 불용성 상태로 고도로 발현되는 녹색형광단백질(이하, 'GFP' 라 칭함.)을 제작하기 위하여 GFP의 아미노산 서열에서 218번째 메티오닌(M218)부터 알라닌(alanine) 위치까지 돌연변이(mutation)를 유발하였다. In order to prepare a green fluorescent protein (hereinafter referred to as 'GFP') highly expressed in an insoluble state, the amino acid sequence of GFP is substituted with 218th methionine (M218) Mutation to the alanine site was induced.
그 후에, 모든 내부 메티오닌(Met) 자리들에서 돌연변이를 유발시켜 메티오닌-결실 GFP(이하, "r5M-GFP" 라 칭함.)를 유도하였다. Thereafter, a mutation was induced in all internal methionine (Met) sites to derive methionine-deleted GFP (hereinafter referred to as "r5M-GFP").
상기 돌연변이가 유발된 r5M-GFP 아미노산 서열에서 172번과 173번 사이에 항균 펩타이드 삽입자리를 만들기 위하여 제한효소 KpnI 및 BamHI 으로 절단하였고, 절단된 r5M-GFP construct 를 주형으로 하여 이를 증폭시키기 위해 증폭에 필요한 프라이머(primer)를 설계하여 중합효소 연쇄반응법(Polymerase Chain Reaction, PCR)을 실시하였다. The mutant-induced r5M-GFP amino acid sequence was cleaved with restriction enzymes KpnI and BamHI to create an antimicrobial peptide insertion site between positions 172 and 173, amplified using the truncated r5M-GFP construct as a template and amplified The required primers were designed and polymerase chain reaction (PCR) was performed.
또한, PG1을 코딩하는 유전자 및 PMAP36을 코딩하는 유전자도 r5M-GFP에 삽입될 부분을 KpnI 및 BamHI 으로 절단 후, 절단된 DNA 단편을 주형으로 하여 증폭에 필요한 프라이머(primer)를 설계한 후, 중합효소 연쇄반응법(Polymerase Chain Reaction, PCR)을 실시하였다. In addition, the gene coding for PG1 and the gene coding for PMAP36 also were cleaved with KpnI and BamHI at the site to be inserted into r5M-GFP, and a primer necessary for amplification was designed using the DNA fragment as a template, And the polymerase chain reaction (PCR) was performed.
상기 과정에서 증폭된 DNA 단편을 라이게이션(ligation) 시켜 이들을 재조합하였는데, 항균 펩타이드 PG1 과 PMAP36 유전자는 GFP 의 아미노산 서열에서 172번과 173번 사이 루프(loop)지역으로 삽입된다. (상기 GFP에 항균 펩타이드가 결합된 구조를 “r5M-172AMP173” 로 표기). The DNA fragments amplified in the above procedure were ligated and recombined. The antimicrobial peptides PG1 and PMAP36 were inserted into the loop region between positions 172 and 173 in the amino acid sequence of GFP. (&Quot; r5M-172AMP173 " represents the structure in which the antimicrobial peptide is bound to GFP).
상기 재조합된 유전자(r5-M172AMP173)는 pET30b 발현벡터의 NdeI과 XhoI 제한효소 자리로 삽입되는데, 이 때 r5M-172AMP173 의 양 말단에 히스티틴 태그(his-tag)가 부착되도록 상기 PCR 프라이머 설계시 his-tag 서열을 추가한다. his-tag는 재조합 단백질 말단에 붙힌 여러 개의 histidine를 가리키며, histidine의 금속이온에 대한 선택적 결합능을 이용하면 재조합 단백질을 쉽게 분리·정제할 수 있다. The recombinant gene (r5-M172AMP173) is inserted into the NdeI and XhoI restriction sites of the pET30b expression vector. When the His-tag is attached to both ends of r5M-172AMP173, Add the -tag sequence. His-tag refers to several histidine attached to the end of the recombinant protein, and the selective binding ability of histidine to the metal ion can be used to easily separate and purify the recombinant protein.
histidine은 이미다졸기를 가진 아미노산으로 주로 Ni2+또는 Fe2+ 등의 2가 금속이온과 결합을 잘한다. his-tag 는 재조합 단백질의 발현벡터를 만드는 단계에서부터 부착 여부를 고려해야 하는데, 재조합 단백질 염기서열의 앞부분이나 뒷부분에 여러 개의 histidine이 발현되도록 하는데, 보통 발현시키는 histidine의 개수는 6개 이상이다. Histidine is an amino acid with an imidazole group and binds mainly with divalent metal ions such as Ni 2+ or Fe 2+ . His-tag should consider adhesion from the step of making the expression vector of recombinant protein. The number of histidine to be expressed is more than 6, so that several histidine is expressed in the front part or the rear part of the recombinant protein base sequence.
상기 과정을 통하여 r5M-172PG1173 유전자가 삽입된 pET30b 발현벡터및 r5M-172PMAP36173 유전자가 삽입된 pET30b 발현벡터가 제작되었고, 이들 각각은 대장균에 도입되어 대장균의 형질전환이 유도되었다. 본 발명에서 사용한 대장균은 E.coli BL21 이나 이에 한정되지 아니한다. Through the above procedure, a pET30b expression vector in which the r5M-172PG1173 gene was inserted and a pET30b expression vector in which the r5M-172PMAP36173 gene was inserted were constructed, and each of them was introduced into E. coli and the transformation of E. coli was induced. E. coli BL21 used in the present invention is not limited to E. coli BL21.
상기 형질전환된 E. coli BL21은 37℃의 1L LB 배지(Luria-Bertani broth)에서 배양되었고, 0.1mM IPTG(Isopropyl β-D thiogalactoside)를 투입하여 단백질의 발현을 유도하였다. 이후, 원심분리기를 이용하여 세포를 수거하였다. The transformed E. coli BL21 was cultured in 1 L LB medium (Luria-Bertani broth) at 37 ° C and 0.1 mM IPTG (Isopropyl β-D thiogalactoside) was added to induce protein expression. Then, the cells were harvested using a centrifuge.
수거된 세포에서 불용성의 분획물을 추출하기 위하여 초음파 분쇄기(sonicator)를 이용하여 세포를 파쇄하였고, 파쇄된 용해물(lysate)을 원심분리 하여 가용성 및 불용성 분획물을 수득하였다. 불용성 분획물은 용해버퍼(lysis buffer)로 재부유 되었고, 여기에 라이소자임(lysozyme)을 투입하여 세포벽을 파괴하였고, DNA 분해효소(DNase)를 투입하여 DNA를 절단한 후, 원심분리하여 응집체(inclusion bodies) 형태로 발현된 불용성 단백질을 분리하였다. To extract insoluble fractions from the collected cells, the cells were disrupted using an ultrasonic sonicator, and the lysate was centrifuged to obtain soluble and insoluble fractions. The insoluble fractions were resuspended in a lysis buffer, lysozyme was added thereto to destroy the cell wall, DNA was digested by adding DNAase (DNase), and the DNA was cut and centrifuged to remove inclusion bodies ) Were isolated.
상기 분리된 불용성 단백질의 수율을 높이기 위하여 라이소자임과 DNA 분해효소를 포함하는 sodium phosphate buffer 를 이용하여 cell wall, cell debris, gDNA 를 제거하는 세척 단계(washing step)를 2회 실시하였다. In order to increase the yield of the separated insoluble protein, a washing step of removing cell wall, cell debris and gDNA was performed twice using sodium phosphate buffer containing lysozyme and DNA degrading enzyme.
불용성 단백질은 상기와 같이 응집체 형태로 발현됨으로써, 항균 펩타이드가 독성으로 작용하여 숙주세포의 성장을 저해하는 것을 극소화할 수 있게 되고, 또한 항균 펩타이드와 같이 분자량이 작은 크기의 폴리펩타이드의 경우 숙주세포 내에 존재하는 단백질 분해효소에 의해 분해되기 쉬운데 이 또한 막을 수 있어 항균 펩타이드의 제조과정에서 생산 수율을 높일 수 있다. The insoluble protein is expressed in the aggregate form as described above, so that the antimicrobial peptide acts as a toxic substance to minimize the inhibition of the growth of the host cell. In the case of a polypeptide having a small molecular weight such as an antimicrobial peptide, It is easy to be decomposed by an existing protease, and this can also be blocked, which can increase the production yield in the course of producing the antimicrobial peptide.
상기 응집체 형태로 발현된 불용성 단백질은 니켈-니트로트리아세트산 컬럼 크로마토그래피(Ni-NTA column chromatograph)에 의해서 정제되고, 용출된 분획(elution fraction)은 전기영동(SDS-PAGE)에 의해서 분석되고, 탈이온수를 투석액으로 하여 투석된다. 투석은 정제과정에서 투입된 우레아, 이미다졸, NaCl 을 제거하기 위한 것으로 이 과정을 거친 후, 불용성 단백질(r5M-172PG1173, r5M-172PMAP36173)을 동결건조 시키는데, 동결건조를 통하여 불필요한 물을 제거하게 된다. 이와 같이 투석 및 동결건조 과정을 거쳐 고농도의 불용성 단백질을 수득하게 된다. The insoluble protein expressed in the form of aggregate was purified by a Ni-NTA column chromatograph, and the elution fraction eluted was analyzed by electrophoresis (SDS-PAGE) Dialyzed with ionized water as a dialysis solution. Dialysis is a process for removing urea, imidazole and NaCl in the purification process. After this process, the insoluble protein (r5M-172PG1173, r5M-172PMAP36173) is lyophilized, and free water is removed through freeze-drying. Thus, a high concentration of insoluble protein is obtained through a dialysis and lyophilization process.
상기 동결건조 된 불용성 단백질은 시안화브롬(CNBr) 처리를 위해 상기 70% 포름산(formic acid)의 투입으로 용해된다. 불용성 단백질에 시안화브롬이 첨가되면 항균 펩타이드(PG1, PMAP36)의 양측에 위치한 GFP 의 N-말단과 C-말단 부위가 잘리고 항균 펩타이드만을 수득하게 된다. 이후, 항균 펩타이드는 역상 고압액체크로마토그래피(reverse phase HPLC)에 의해 정제된다. The lyophilized insoluble protein is dissolved by the addition of 70% formic acid for treatment with cyanide bromide (CNBr). When bromine cyanide is added to the insoluble protein, the N-terminal and C-terminal regions of GFP located on both sides of the antimicrobial peptide (PG1, PMAP36) are cleaved and only the antimicrobial peptide is obtained. The antimicrobial peptide is then purified by reverse phase high pressure liquid chromatography (reverse phase HPLC).
정제된 항균 펩타이드는 refolding buffer를 첨가하여 자연(native)상태로 재접힘 되고 동시에 투석에 의해서 refolding buffer가 탈이온수로 교체 된다. The purified antimicrobial peptide is refolded to the native state by adding a refolding buffer, and at the same time, the refolding buffer is replaced with deionized water by dialysis.
정제된 항균 펩타이드 PG1 및 PMAP36의 항균활성을 확인하기 위해서 E.coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 및 Staphylococcus aureus ATCC 29213 을 사용하여 spot-on-lawn test 를 수행한 결과, 항균 활성을 보임을 확인하였고, 그 결과는 표 1 과 같다. In order to confirm the antibacterial activities of the purified antimicrobial peptides PG1 and PMAP36, spot-on-lawn test was conducted using E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213, , And the results are shown in Table 1.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예 1. 불용성 녹색형광단백질과 항균 펩타이드가 결합된 융합단백질의 제조를 위한 DNA 증폭 및 발현벡터의 제조EXAMPLE 1 DNA Amplification and Preparation of an Expression Vector for Preparation of a Fusion Protein Bonded with an Insoluble Green Fluorescent Protein and an Antibacterial Peptide
불용성 녹색형광단백질과 항균 펩타이드가 결합된 융합단백질을 제조하기 위하여 먼저, 불용성 형태의 고도로 발현가능한 GFP를 제작하였다. 이를 위해 GFP의 아미노산 서열에서 218번째 메티오닌(M218)부터 알라닌(alanine) 위치까지 돌연변이(mutation)을 유발하였고, 다음으로, 상기 GFP 내부의 모든 메티오닌(Met) 자리들을 내부 메티오닌-결실 GFP(r5M-GFP)로 만들기 위해서 돌연변이를 유발하였다. In order to prepare a fusion protein comprising an insoluble green fluorescent protein and an antibacterial peptide, a highly expressible GFP of insoluble form was first prepared. For this, a mutation was induced from the 218th methionine (M218) to the alanine site in the amino acid sequence of GFP. Next, all the methionine (Met) sites in the GFP were substituted with the internal methionine-deleted GFP (r5M- GFP). ≪ / RTI >
상기 돌연변이가 유발된 r5M-GFP 아미노산 서열에서 172번과 173번 사이에 항균 펩타이드 삽입자리를 만들기 위하여 제한효소 KpnI 및 BamHI 으로 절단하였고, 절단된 r5M-GFP construct 를 주형으로 하여 이를 증폭시키기 위해 증폭에 필요한 프라이머(primer)를 설계하여 중합효소 연쇄반응법(Polymerase Chain Reaction, PCR)을 실시하였다. 증폭에 필요한 각각의 프라이머 서열은 표 2와 같다. The mutant-induced r5M-GFP amino acid sequence was cleaved with restriction enzymes KpnI and BamHI to create an antimicrobial peptide insertion site between positions 172 and 173, amplified using the truncated r5M-GFP construct as a template and amplified The required primers were designed and polymerase chain reaction (PCR) was performed. Each primer sequence required for amplification is shown in Table 2.
또한, PG1을 코딩하는 유전자(하기 서열번호 1) 및 PMAP36(하기 서열번호 2)을 코딩하는 유전자도 r5M-GFP에 삽입될 부분을 KpnI 및 BamHI 으로 절단 후, 절단된 DNA 단편을 주형으로 하여 증폭에 필요한 프라이머(primer)를 설계한 후, 중합효소 연쇄반응법(Polymerase Chain Reaction, PCR)을 실시하였다. 증폭에 필요한 각각의 프라이머 서열은 표 2와 같다.In addition, the gene encoding PG1 (SEQ ID NO: 1) and PMAP36 (SEQ ID NO: 2 below) were also digested with KpnI and BamHI to insert in r5M-GFP, And then polymerase chain reaction (PCR) was carried out. Each primer sequence required for amplification is shown in Table 2.
서열번호 1; SEQ ID NO: 1;
5'-AGGGGAGGTCGCCTGTGCTATTGTAGGCGTAGGTTCTGCGTCTGTGTCGGACGAGGA-3'5'-AGGGGAGGTCGCCTGTGCTATTGTAGGCGTAGGTTCTGCGTCTGTGTCGGACGAGGA-3 '
서열번호 2;SEQ ID NO: 2;
5-'GGACGATTTAGACGGTTGCGTAAGAAGACCCGAAAACGTTTGAAGAAGATCGGGAAGGT5-'GGACGATTTAGACGGTTGCGTAAGAAGACCCGAAAACGTTTGAAGAAGATCGGGAAGGT
TTTGAAGTGGATTCCTCCCATTGTCGGCTCAATACCCTTGGGTTGTGGG-3'TTTGAAGTGGATTCCTCCCATTGTCGGCTCAATACCCTTGGGTTGTGGG-3 '
상기 프라이머 서열에서 서열번호 3의 염기서열에는 이후 발현벡터 pET30b 와의 결합을 위하여 제한효소 NdeI 인식 서열과 Ni-NTA 컬럼 크로마토 그래피를 위한 6x His tag 서열(5′- CATCACCATCATCACCAT-3′)이 포함되어 있고, 이후 서열번호 4의 염기서열과 서열번호 5의 염기서열에는 시안화브롬 처리를 통한 펩타이드 절단을 위하여 메티오닌을 암호화하는 ATG 코돈이 포함되어 있다. 또한 서열번호 6의 염기서열에는 이후 발현벡터 pET30b 와의 결합을 위하여 제한효소 XhoI의 인식 서열이 포함되어 있고, 서열번호 7(annealing temperature:57℃)와 서열번호 9(annealing temperature:50℃)의 염기서열에는 제한효소 KpnI과 시안화브롬 처리를 통한 펩타이드 절단을 위한 ATG 코돈이 포함되어 있다. 또한, 서열번호 8과 서열번호 10의 염기서열에는 제한효소 BamHI의 인식 서열과 시안화브롬 처리를 통한 펩타이드 절단을 위한 ATG 코돈이 포함되어 있는 것을 특징으로 한다. 상기와 같이 메티오닌을 암호화하는 ATG 코돈을 넣어줄 경우, 차후 융합단백질에서 항균 펩타이드 PG1 과 PMAP36 만을 분리하는 단계에서, 시안화브롬(CNBr) 처리시 메티오닌 잔기 바로 뒤의 펩타이드 결합이 절단되도록 하여 항균 펩타이드만을 분리할 수 있다. In the above primer sequence, the nucleotide sequence of SEQ ID NO: 3 contains a restriction enzyme NdeI recognition sequence and a 6x His tag sequence (5'-CATCACCATCATCACCAT-3 ') for Ni-NTA column chromatography for binding with the expression vector pET30b , And the nucleotide sequence of SEQ ID NO: 4 and the nucleotide sequence of SEQ ID NO: 5 contain an ATG codon which encodes methionine for cleavage of the peptide by cyanide bromination. Also, the nucleotide sequence of SEQ ID NO: 6 contains the recognition sequence of the restriction enzyme XhoI for binding to the expression vector pET30b, and the nucleotide sequence of SEQ ID NO: 7 (annealing temperature: 57 DEG C) and annealing temperature The sequence contains the restriction enzyme KpnI and the ATG codon for peptide cleavage through cyanide bromination. In addition, the nucleotide sequence of SEQ ID NO: 8 and SEQ ID NO: 10 is characterized in that the recognition sequence of BamHI for restriction enzyme and the ATG codon for peptide cleavage through cyanated bromine treatment are contained. When the ATG codon encoding methionine is added as described above, in the step of separating only the antimicrobial peptides PG1 and PMAP36 from the subsequent fusion protein, the peptide bond immediately after the methionine residue is cleaved during treatment with cyanide bromide (CNBr) Can be separated.
상기 과정에서 증폭된 DNA 단편을 라이게이션(ligation) 시켜 이들을 재조합하였는데, 항균 펩타이드 PG1 과 PMAP36 유전자는 GFP 의 아미노산 서열에서 172번과 173번 사이 루프(loop)지역으로 삽입된다. The DNA fragments amplified in the above procedure were ligated and recombined. The antimicrobial peptides PG1 and PMAP36 were inserted into the loop region between positions 172 and 173 in the amino acid sequence of GFP.
상기 과정에 의하여 재조합된 유전자는 “r5M-172PG1173”(항균 펩타이드 PG1 유전자 삽입) 및 “r5M-172PMAP36173”(항균 펩타이드 PMAP36 삽입) 으로 표기한다. 상기 재조합 유전자 r5M-172PG1173의 염기서열은 서열번호 11 이고, r5M-172PMAP36173 의 염기서열은 서열번호 12 로 하기와 같다.The recombinant gene was expressed by "r5M-172PG1173" (insertion of antibiotic peptide PG1 gene) and "r5M-172PMAP36173" (insertion of antimicrobial peptide PMAP36). The nucleotide sequence of the recombinant gene r5M-172PG1173 is SEQ ID NO: 11, and the nucleotide sequence of r5M-172PMAP36173 is SEQ ID NO: 12.
서열번호 11( 5′-> 3′); SEQ ID NO: 11 (5 '-> 3');
CATATGCATCACCATCATCACCATCAGAGCAAAGGCGAAGAACTGTTTACCGGCGTGGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGCCATAAATTTAGCGTGCGTGGCGAAGGCGAAGGCGATGCGACCAACGGCAAACTGACCCTGAAATTTATTTGCACCACCGGTAAACTGCCGGTGCCGTGGCCGACCCTGGTGACCACCCTGGGTTATGGTGTGCAGTGCTTTGCACGTTATCCGGATCACATCAAACGTCATGATTTCTTTAAAAGCGCGCTGCCGGAAGGCTATGTGCAGGAACGTACCATTAGCTTTAAAGATGATGGCACCTATAAAACCCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTGAACCGTATTGAACTGAAAGGCATTGATTTTAAAGAAGATGGCAACATTCTGGGCCATAAACTGGAATATAACTTTAACAGCCATAAAGTGTATATTACCGCGGATAAACAGAAAAACGGCATTAAAGCGAACTTTAAAATTCGTCATAACGTGGAAGGTGGTTCTGGTACCATGAGGGGAGGTCGCCTGTGCTATTGTAGGCGTAGGTTCTGCGTCTGTGTCGGACGAGGAATGGGATCCGGTGGCGATGGCAGCGTGCAGCTGGCGGATCATTATCAGCAGAACACCCCGATTGGCGATGATAACCATTATCTGAGCACCCAGAGCGTGCTGCTGAAAGATCCGAACGAAAAACGTGATCACGCGGTGCTGCTGGAATTTGTGACCGCGGCGGGCATTACCCACGGCAAAGATGAACTGTATAAACATCACCATCATCACCATTAATAACTCGAGATC
CATATGCATCACCATCATCACCATCAGAGCAAAGGCGAAGAACTGTTTACCGGCGTGGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGCCATAAATTTAGCGTGCGTGGCGAAGGCGAAGGCGATGCGACCAACGGCAAACTGACCCTGAAATTTATTTGCACCACCGGTAAACTGCCGGTGCCGTGGCCGACCCTGGTGACCACCCTGGGTTATGGTGTGCAGTGCTTTGCACGTTATCCGGATCACATCAAACGTCATGATTTCTTTAAAAGCGCGCTGCCGGAAGGCTATGTGCAGGAACGTACCATTAGCTTTAAAGATGATGGCACCTATAAAACCCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTGAACCGTATTGAACTGAAAGGCATTGATTTTAAAGAAGATGGCAACATTCTGGGCCATAAACTGGAATATAACTTTAACAGCCATAAAGTGTATATTACCGCGGATAAACAGAAAAACGGCATTAAAGCGAACTTTAAAATTCGTCATAACGTGGAAGGTGGTTCTGGTACCATGAGGGGAGGTCGCCTGTGCTATTGTAGGCGTAGGTTCTGCGTCTGTGTCGGACGAGGAATGGGATCCGGTGGCGATGGCAGCGTGCAGCTGGCGGATCATTATCAGCAGAACACCCCGATTGGCGATGATAACCATTATCTGAGCACCCAGAGCGTGCTGCTGAAAGATCCGAACGAAAAACGTGATCACGCGGTGCTGCTGGAATTTGTGACCGCGGCGGGCATTACCCACGGCAAAGATGAACTGTATAAACATCACCATCATCACCATTAATAACTCGAGATC
서열번호 12( 5′-> 3′);SEQ ID NO: 12 (5 '-> 3');
CATATGCATCACCATCATCACCATCAGAGCAAAGGCGAAGAACTGTTTACCGGCGTGGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGCCATAAATTTAGCGTGCGTGGCGAAGGCGAAGGCGATGCGACCAACGGCAAACTGACCCTGAAATTTATTTGCACCACCGGTAAACTGCCGGTGCCGTGGCCGACCCTGGTGACCACCCTGGGTTATGGTGTGCAGTGCTTTGCACGTTATCCGGATCACATCAAACGTCATGATTTCTTTAAAAGCGCGCTGCCGGAAGGCTATGTGCAGGAACGTACCATTAGCTTTAAAGATGATGGCACCTATAAAACCCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTGAACCGTATTGAACTGAAAGGCATTGATTTTAAAGAAGATGGCAACATTCTGGGCCATAAACTGGAATATAACTTTAACAGCCATAAAGTGTATATTACCGCGGATAAACAGAAAAACGGCATTAAAGCGAACTTTAAAATTCGTCATAACGTGGAAGGTGGTTCTGGTACCATGGGACGATTTAGACGGTTGCGTAAGAAGACCCGAAAACGTTTGAAGAAGATCGGGAAGGTTTTGAAGTGGATTCCTCCCATTGTCGGCTCAATACCCTTGGGTTGTGGGATGGGATCCGGTGGCGATGGCAGCGTGCAGCTGGCGGATCATTATCAGCAGAACACCCCGATTGGCGATGATAACCATTATCTGAGCACCCAGAGCGTGCTGCTGAAAGATCCGAACGAAAAACGTGATCACGCGGTGCTGCTGGAATTTGTGACCGCGGCGGGCATTACCCACGGCAAAGATGAACTGTATAAACATCACCATCATCACCATTAATAACTCGAG
CATATGCATCACCATCATCACCATCAGAGCAAAGGCGAAGAACTGTTTACCGGCGTGGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGCCATAAATTTAGCGTGCGTGGCGAAGGCGAAGGCGATGCGACCAACGGCAAACTGACCCTGAAATTTATTTGCACCACCGGTAAACTGCCGGTGCCGTGGCCGACCCTGGTGACCACCCTGGGTTATGGTGTGCAGTGCTTTGCACGTTATCCGGATCACATCAAACGTCATGATTTCTTTAAAAGCGCGCTGCCGGAAGGCTATGTGCAGGAACGTACCATTAGCTTTAAAGATGATGGCACCTATAAAACCCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTGAACCGTATTGAACTGAAAGGCATTGATTTTAAAGAAGATGGCAACATTCTGGGCCATAAACTGGAATATAACTTTAACAGCCATAAAGTGTATATTACCGCGGATAAACAGAAAAACGGCATTAAAGCGAACTTTAAAATTCGTCATAACGTGGAAGGTGGTTCTGGTACCATGGGACGATTTAGACGGTTGCGTAAGAAGACCCGAAAACGTTTGAAGAAGATCGGGAAGGTTTTGAAGTGGATTCCTCCCATTGTCGGCTCAATACCCTTGGGTTGTGGGATGGGATCCGGTGGCGATGGCAGCGTGCAGCTGGCGGATCATTATCAGCAGAACACCCCGATTGGCGATGATAACCATTATCTGAGCACCCAGAGCGTGCTGCTGAAAGATCCGAACGAAAAACGTGATCACGCGGTGCTGCTGGAATTTGTGACCGCGGCGGGCATTACCCACGGCAAAGATGAACTGTATAAACATCACCATCATCACCATTAATAACTCGAG
상기 서열번호 11과 서열번호 12의 염기서열에는 하기 서열번호 13 및 서열번호 14 의 염기서열로 이루어진 링커(linker)가 포함되어 있는 것을 특징으로 한다.The nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12 includes a linker comprising the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14.
서열번호 13;SEQ ID NO: 13;
5′- GGTGGTTCT - 3′5'-GGTGGTTCT-3 '
서열번호 14;SEQ ID NO: 14;
5′- GGATCCGGTGGC - 3′5'-GGATCCGGTGGC-3 '
상기 r5M-172PG1173과 r5M-172PMAP36173 은 pET30b 발현벡터의 NdeI과 XhoI 제한효소 자리로 삽입되어 재조합 발현벡터가 제조된다.The r5M-172PG1173 and r5M-172PMAP36173 are inserted into the NdeI and XhoI restriction sites of the pET30b expression vector to produce a recombinant expression vector.
실시예 2. 대장균 형질전환체의 배양 및 융합단백질의 발현 유도Example 2 Culture of Escherichia coli Transformants and Induction of Fusion Protein Expression
상기 실시예 1.에서 얻어진 발현벡터 pET30b를 E.coil BL21에 도입시킨 후, 37℃의 1L LB 배지(Luria-Bertani broth)에서 배양항고, 0.1mM IPTG(Isopropyl β-D thiogalactoside)를 투입하여 단백질의 발현을 유도하였다. 단백질의 발현은 배양물의 혼탁도가 OD600(Optical Density 600nm) 즉, 빛 파장의 600nm에서의 밀도값이 0.6 내지 0.8 에 이를 때까지 유도되었고, 이후, 5시간 더 발현이 유도되었다. The expression vector pET30b obtained in Example 1 was introduced into E. coli BL21 and cultured in 1 L LB medium (Luria-Bertani broth) at 37 ° C, and 0.1 mM IPTG (Isopropyl β-D thiogalactoside) . Protein expression was induced until the turbidity of the culture reached OD600 (Optical Density 600 nm), that is, the density value at 600 nm of the light wavelength was 0.6 to 0.8, and then the expression was induced for further 5 hours.
실시예 3. 불용성 융합단백질의 분리 Example 3 Isolation of Insoluble Fusion Proteins
상기 실시예 2.에서 발현이 유도된 배양물을 4℃, 8000rpm 조건에서 10분 동안 원심분리하여 대장균 세포를 회수하였다. 수거된 세포는 초음파 분쇄기(sonicator)에 의해서 파쇄되었고, 파쇄된 세포 파쇄물은 4℃,13000rpm 조건에서 20분 동안 원심분리하여 가용성 분획물과 불용성 단백질이 포함된 침전물(pellet)을 수득하였다. 불용성 침전물은 1L당 40ml의 용해버퍼(100mM sodium chloride 를 포함하는 pH 7.4의 20mM sodium phosphate buffer, 0.5% triton-X100, 0.1mM PMSF, 1mM DTT)로 재부유(resuspend)시켰다. 이후, 상기 용해물에 라이소자임(0.1mg/ml)과 DNA 분해효소(0.01mg/ml)를 넣고 상온에서 20분 동안 incubation 하였다. 이 과정에서 라이소자임에 의해 세포벽이 파괴되고, DNA 분해효소에 의해서 DNA가 절단된다. The culture in which the expression was induced in Example 2 was centrifuged at 4 ° C and 8,000 rpm for 10 minutes to collect E. coli cells. The collected cells were disrupted by an ultrasonic sonicator and the disrupted cell lysates were centrifuged at 4 ° C and 13000 rpm for 20 minutes to obtain pellets containing soluble fractions and insoluble proteins. The insoluble precipitate was resuspended in 40 ml of lysis buffer (20 mM sodium phosphate buffer, 0.5% triton-X100, 0.1 mM PMSF, 1 mM DTT, pH 7.4, containing 100 mM sodium chloride) per liter. Then, lysozyme (0.1 mg / ml) and DNA-degrading enzyme (0.01 mg / ml) were added to the lysate and incubated at room temperature for 20 minutes. In this process, the cell wall is destroyed by lysozyme and the DNA is cleaved by the DNA-degrading enzyme.
이후, 상기 라이소자임과 DNA분해효소가 첨가된 용해물을 4℃, 8000rpm 조건에서 10분간 원심분리하여 응집체(inclusion bodies) 형태로 발현된 불용성 단백질을 수득하였다. 수득된 불용성 단백질의 수율을 높이기 위하여 라이소자임과 DNA 분해효소를 포함하는 sodium phosphate buffer 를 이용하여 cell wall, cell debris, gDNA 를 제거하는 세척 단계(washing step)를 2회 실시하였다. Thereafter, the lysozyme and the DNA degrading enzyme-added lysate were centrifuged at 4 ° C and 8,000 rpm for 10 minutes to obtain insoluble proteins expressed as inclusion bodies. In order to increase the yield of the insoluble protein, washing step was performed twice to remove cell wall, cell debris and gDNA by using sodium phosphate buffer containing lysozyme and DNA degrading enzyme.
다음으로, 상기 불용성 단백질은 8M urea 와 30mM imidazole 을 포함하는 pH7.4의 20mM sodium phosphate buffer 로 재부유시켰고, Ni-NTA 컬럼 크로마토그래피(GE Healthcare Bio-Sciences, Sweden)에 의해서 정제되었다. Next, the insoluble protein was resuspended in 20 mM sodium phosphate buffer (pH 7.4) containing 8 M urea and 30 mM imidazole, and purified by Ni-NTA column chromatography (GE Healthcare Bio-Sciences, Sweden).
정제 후 용출된 분획은 SDS-PAGE에 의해서 분석되었고, 상온에서 탈이온수를 투석액으로 하여 투석 되었다. The fraction eluted after purification was analyzed by SDS-PAGE and dialyzed with deionized water as a dialysis solution at room temperature.
실시예 4. 항균 펩타이드 PG1, PMAP36의 분리 및 정제 Example 4 Isolation and Purification of Antimicrobial Peptides PG1 and PMAP36
상기 실시예 3.에서 정제 후 투석된 불용성 단백질은 동결건조되고, 70% 포르믹산(formic acid)에 의해서 용해된다.After purification in Example 3 above, the dialyzed insoluble protein is lyophilized and dissolved by 70% formic acid.
상기 불용성 단백질 용액에 시안화브롬(CNBr) 용액을 첨가한 후, 암실에서 24시간 동안 incubation 시킴으로써, 항균 펩타이드 PG1과 PMAP36 의 양쪽에 위치한 GFP의 N-말단과 C-말단 부위는 절단되었다. The N-terminal and C-terminal portions of GFP located on both sides of the antimicrobial peptides PG1 and PMAP36 were cleaved by adding the cyanoborate (CNBr) solution to the insoluble protein solution and then incubating in the dark room for 24 hours.
상기 포르믹산과 시안화브롬은 동결건조에 의해서 제거되고, 이후, 역상 고압크로마토그래피를 위한 버퍼에 의해서 용해 된다. The formic acid and bromide cyanide are removed by lyophilization and then dissolved by a buffer for reversed phase high pressure chromatography.
정제된 단백질의 순도는 16% tris-tricine SDS-PAGE 에 의해서 각 단계에서마다 분석되어진다. The purity of the purified protein is analyzed at each step by 16% tris-tricine SDS-PAGE.
항균 펩타이드 PG1 과 PMAP36은 역 역상 고압크로마토그래피(Waters Deltapak C18 column 7.8, 300mm)에 의해서 정제되었는데, 0.1% 트리플루오로아세트산(trifluoroacetic acid)이 포함되고, 아세토니트릴(acetonitrile)이 5 내지 90%까지 직선 구배농도를 지니고 있는 관에 2.5ml/min의 유속으로 1시간동안 시료를 흘려주었다. 흡광도는 220nm와 280nm 둘 다에서 관찰하였고, 일치하는 피크는 16% tris-tricine SDS-PAGE 에 의해서 분석되었다. The antimicrobial peptides PG1 and PMAP36 were purified by reversed phase high pressure chromatography (Waters Deltapak C18 column 7.8, 300 mm) containing 0.1% trifluoroacetic acid and 5 to 90% acetonitrile The sample was flowed at a flow rate of 2.5 ml / min in a tube having a linear gradient concentration for 1 hour. Absorbance was observed at both 220 and 280 nm, and the corresponding peaks were analyzed by 16% tris-tricine SDS-PAGE.
정제된 분획은 refolding condition을 만들어주기 위해 동결건조를 통하여 불필요한 버퍼를 제거하는 과정을 거친 후, 8M 우레아, 5mM 환원된 글루타치온(glutathione) 및 0.5mM 산화된 글루타치온(glutathione)을 포함하는 20mM sodium phosphate buffer(pH7.4)로 구성된 refolding buffer를 첨가하여 자연상태로 refolding 되고, 동시에 투석에 의해서 refolding buffer가 탈이온수로 교체되었다. 이후, 정제 및 투석된 펩타이드는 동결건조 시켰다. The purified fractions were subjected to freeze-drying to remove unnecessary buffers to make a refolding condition. Then, 20 mM sodium phosphate buffer containing 8M urea, 5 mM reduced glutathione and 0.5 mM oxidized glutathione (pH 7.4), and the refolding buffer was replaced with deionized water by dialysis. The purified and dialyzed peptides were then lyophilized.
실험예 1. r5M-172PG1173 유전자가 삽입된 pET30b 발현벡터의 단백질 발현이 유도 및 비유도 된 E.coli BL21의 성장 곡선 측정· 비교Experimental Example 1. Measurement and comparison of growth curve of E. coli BL21 induced and non-induced protein expression of pET30b expression vector in which r5M-172PG1173 gene was inserted
r5M172-PG1-173 유전자가 삽입된 pET30b 발현벡터가 도입된 E.coli BL21에서 r5M172-PG1-173의 단백질 발현이 유도된 것과 유도되지 않은 E.coli BL21 의 성장을 광학 밀도(optical density) 측정을 통하여 비교하였다. The optical density measurement of r5M172-PG1-173 protein induced and uninduced E. coli BL21 expression in E. coli BL21 transfected with the pET30b expression vector inserted with r5M172-PG1-173 gene Respectively.
그 결과, 도 2에서 보는 바와 같이 r5M-172PG1173 단백질 발현은 대장균의 성장률에 크게 영향을 미치지 않음을 알 수 있다. 즉, 지지체로서 불용성 GFP 로 삽입된 항균 펩타이드는 대장균의 성장에 큰 영향을 미치지 않는다. As a result, as shown in FIG. 2, it can be seen that r5M-172PG1173 protein expression did not significantly affect the growth rate of E. coli. That is, the antimicrobial peptide inserted as insoluble GFP as a support does not greatly affect the growth of E. coli.
비록, 항균 펩타이드의 안정적인 생산을 위하여 퓨전 파트너들과 결합시키는 많은 시도들이 있어왔지만, 낮은 생산 수율로 인하여 고영양의 배지 투입,퓨전파트너 사이에 유전자의 복수 카피 삽입 등 추가적인 생산비용이 따르는 보완책이 필요하였다. 그러나 불용성의 상태로 고도로 발현되도록 제작된 GFP에 항균 펩타이드의 삽입만으로 제조된 본 발명의 융합단백질은 숙주세포의 성장 저해가 극소화 되어 높은 수율로 항균 펩타이드를 생산할 수 있다. Although there have been many attempts to associate with fusion partners for the stable production of antimicrobial peptides, low production yields require complementary measures with additional production costs such as high nutrient loading, multiple copies of the gene inserted between fusion partners Respectively. However, the fusion protein of the present invention prepared by inserting an antimicrobial peptide into GFP that is highly expressed in an insoluble state can minimize the growth inhibition of host cells and produce an antimicrobial peptide with a high yield.
실험예 2. r5M-172PG1173 유전자가 삽입된 pET30b 발현벡터의 시간 경과에 따른 단백질 생산성 측정Experimental Example 2. Measurement of Protein Productivity over Time of pET30b Expression Vector Containing r5M-172PG1173 Gene
대장균 내에서 r5M-172PG1173 유전자가 삽입된 pET30b 발현벡터의 시간 경과(3h, 4h, 5h)에 따른 r5M-172PG1173 단백질 생산을 전체 세포 단백질을 샘플로 하여 전기영동(Sodiumdodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE)을 통하여 확인하였다.The production of r5M-172PG1173 protein according to the lapse of time (3h, 4h, 5h) of pET30b expression vector in which r5M-172PG1173 gene was inserted in E. coli was analyzed by SDS-PAGE ).
그 결과, 도 3에서 보는 바와 같이 r5M-172PG1173 단백질 발현이 유도되지 않은 lane(UI)에서와 달리 단백질 발현이 유도된 lane(3h,4h,5h)을 살펴보면 E.coli BL21 내에서 r5M-172PG1173 단백질이 3 내지 5시간에서 모두 뚜렷하게 발현됨을 알 수 있다. As shown in FIG. 3, lane (3h, 4h, 5h) in which protein expression was induced is different from that in lane (UI) in which r5M-172PG1173 protein expression is not induced. Is clearly expressed in 3 to 5 hours.
실험예 3. E.coli BL21에서 도입된 pET30b 발현벡터 내의 r5M-172PG1173 및 r5M-172PMAP36173의 단백질 생산성 측정Experimental Example 3. Measurement of protein productivity of r5M-172PG1173 and r5M-172PMAP36173 in pET30b expression vector introduced in E. coli BL21
r5M-GPF 유전자로 각각 삽입된 PG1 및 PMAP36 유전자는 pET30b 발현벡터로 클로닝 된 후 E.coli BL21에서 5시간 동안 발현이 유도되었다. The PG1 and PMAP36 genes inserted with the r5M-GPF gene were cloned into the pET30b expression vector and then expressed in E. coli BL21 for 5 hours.
상기 재조합 단백질은 세포 내 전체 댄백질의 약 45 내지 50 % 를 구성할 것으로 예상된다. 세포 배양물 1리터 당 1.4g 수득된 r5M-172PG1173 불용성 단백질(31kDa) 과 세포 배양물 1리터 당 1.3g 수득된 r5M-172PG1173 불용성 단백질(33kDa)은 전기영동의 최종 불용성 분획 부분에서 보여지는 바와 같이 최종적으로 목적 단백질의 거의 90%를 차지하였다(도 4a 및 도 4b). The recombinant protein is expected to constitute about 45-50% of total intracellular protein. 1.4 g / l of cell culture The obtained r5M-172PG1173 insoluble protein (31 kDa) and 1.3 g / l of cell culture The obtained r5M-172PG1173 insoluble protein (33 kDa), as shown in the final insoluble fraction fraction of the electrophoresis And finally occupied almost 90% of the target protein (FIGS. 4A and 4B).
상기 불용성 단백질은 Ni-NTA 크로마토그래피에 의해서 정제되어 지고, 정제된 목적 단백질은 풀(pool)화 하였고, 탈이온수를 투석액으로 하여 투석되었다. The insoluble protein was purified by Ni-NTA chromatography, and the purified target protein was pooled and dialyzed with deionized water as a dialysis liquid.
실험예 4. 항균 펩타이드 PG1 및 PMAP36의 수율 측정Experimental Example 4. Measurement of yield of antimicrobial peptides PG1 and PMAP36
상기 실험예 3.에서 투석된 단백질 샘플은 동결건조 되고, 포르믹산(formic acids)과 함께 시안화브롬(CNBr) 처리 되었다. 그 결과, r5M-GPF로부터 PG1과 PMAP36은 각각 절단 되었고, 이후, PG1(2.4kDa) 및 PMAP36(4.2kDa)은 HPLC에 의하여 정제된 후 그 수율을 측정한 결과, 표 3에서 보는 바와 같이 두 단백질 모두 최대 약 12mg 의 수율을 보였다. The protein sample dialyzed in Experimental Example 3 was lyophilized and treated with cyanide bromide (CNBr) together with formic acids. As a result, PG1 and PMAP36 were cleaved from r5M-GPF, respectively. PG1 (2.4 kDa) and PMAP36 (4.2 kDa) were purified by HPLC and their yields were measured. As a result, All showed a maximum yield of about 12 mg.
실험예 5. 항균 펩타이드 PG1 및 PMAP36의 순도 분석Experimental Example 5. Purity analysis of antimicrobial peptides PG1 and PMAP36
순도 높은 PG1 및 PMAP36을 얻기 위하여 tris-tricine SDS-PAGE 로 단백질 정제의 각 단계 마다 추출되는 단백질 샘플을 로딩(laoding) 후 전기영동하여 순도를 분석하여 제조과정을 진행한 결과 95% 이상의 순도를 나타내는 항균펩타이드 PG1 및 PMAP36을 수득하였다(도 5a 및 5b). In order to obtain high-purity PG1 and PMAP36, the protein samples extracted at each step of protein purification by tris-tricine SDS-PAGE were laid and electrophoretically analyzed for purity. As a result, the purity was 95% or more Antimicrobial peptides PG1 and PMAP36 were obtained (Figures 5a and 5b).
또한, 정제된 PG1은 항-PG1 항체를 사용하여 웨스턴 블롯(western blot)으로 확인하였다. 도 5c에서 보여지는 바와 같이 2.4kDa 위치에서 PG1이 뚜렷하게 확인되었다. In addition, purified PG1 was confirmed by western blot using anti-PG1 antibody. PG1 was clearly identified at the 2.4kDa position as shown in Figure 5C.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다.
Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> University-Industry Cooperation Foundation, Konkuk University <120> METHOD FOR PRODUCING ANTIMICROBIAL PEPTIDE USING INSOLUBLE GREEN FLUORESCENT PROTEIN AS A SCAFFOLD <130> P14-E610 <160> 14 <170> KopatentIn 2.0 <210> 1 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding Protegrin1 (PG1) <400> 1 aggggaggtc gcctgtgcta ttgtaggcgt aggttctgcg tctgtgtcgg acgagga 57 <210> 2 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding Pig myeloid antibacterial peptide36 (PMAP36) <400> 2 ggacgattta gacggttgcg taagaagacc cgaaaacgtt tgaagaagat cgggaaggtt 60 ttgaagtgga ttcctcccat tgtcggctca atacccttgg gttgtggg 108 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 catatgcatc accatcatca ccatcagagc 30 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 catggtacca gaaccacctt ccacgttatg ac 32 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 atgggatccg gtggcgatgg cagcgt 26 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 gtggtggtgc tcgagttatt aatggtg 27 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 gcggttctgg tggtaccatg aggggaggtc gcctgtg 37 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 gccaccggat cccattcctc gtccgacaca gacg 34 <210> 9 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 gcggttctgg tggtaccatg ggacgattta gacggttg 38 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 caccggatcc catcccacaa cccaagggta 30 <210> 11 <211> 843 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding recombinant protein of GFP and PG1 <400> 11 catatgcatc accatcatca ccatcagagc aaaggcgaag aactgtttac cggcgtggtg 60 ccgattctgg tggaactgga tggcgatgtg aacggccata aatttagcgt gcgtggcgaa 120 ggcgaaggcg atgcgaccaa cggcaaactg accctgaaat ttatttgcac caccggtaaa 180 ctgccggtgc cgtggccgac cctggtgacc accctgggtt atggtgtgca gtgctttgca 240 cgttatccgg atcacatcaa acgtcatgat ttctttaaaa gcgcgctgcc ggaaggctat 300 gtgcaggaac gtaccattag ctttaaagat gatggcacct ataaaacccg tgcggaagtg 360 aaatttgaag gcgataccct ggtgaaccgt attgaactga aaggcattga ttttaaagaa 420 gatggcaaca ttctgggcca taaactggaa tataacttta acagccataa agtgtatatt 480 accgcggata aacagaaaaa cggcattaaa gcgaacttta aaattcgtca taacgtggaa 540 ggtggttctg gtaccatgag gggaggtcgc ctgtgctatt gtaggcgtag gttctgcgtc 600 tgtgtcggac gaggaatggg atccggtggc gatggcagcg tgcagctggc ggatcattat 660 cagcagaaca ccccgattgg cgatgataac cattatctga gcacccagag cgtgctgctg 720 aaagatccga acgaaaaacg tgatcacgcg gtgctgctgg aatttgtgac cgcggcgggc 780 attacccacg gcaaagatga actgtataaa catcaccatc atcaccatta ataactcgag 840 atc 843 <210> 12 <211> 891 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding recombinant protein of GFP and PMAP36 <400> 12 catatgcatc accatcatca ccatcagagc aaaggcgaag aactgtttac cggcgtggtg 60 ccgattctgg tggaactgga tggcgatgtg aacggccata aatttagcgt gcgtggcgaa 120 ggcgaaggcg atgcgaccaa cggcaaactg accctgaaat ttatttgcac caccggtaaa 180 ctgccggtgc cgtggccgac cctggtgacc accctgggtt atggtgtgca gtgctttgca 240 cgttatccgg atcacatcaa acgtcatgat ttctttaaaa gcgcgctgcc ggaaggctat 300 gtgcaggaac gtaccattag ctttaaagat gatggcacct ataaaacccg tgcggaagtg 360 aaatttgaag gcgataccct ggtgaaccgt attgaactga aaggcattga ttttaaagaa 420 gatggcaaca ttctgggcca taaactggaa tataacttta acagccataa agtgtatatt 480 accgcggata aacagaaaaa cggcattaaa gcgaacttta aaattcgtca taacgtggaa 540 ggtggttctg gtaccatggg acgatttaga cggttgcgta agaagacccg aaaacgtttg 600 aagaagatcg ggaaggtttt gaagtggatt cctcccattg tcggctcaat acccttgggt 660 tgtgggatgg gatccggtgg cgatggcagc gtgcagctgg cggatcatta tcagcagaac 720 accccgattg gcgatgataa ccattatctg agcacccaga gcgtgctgct gaaagatccg 780 aacgaaaaac gtgatcacgc ggtgctgctg gaatttgtga ccgcggcggg cattacccac 840 ggcaaagatg aactgtataa acaccatcac caccatcact aataactcga g 891 <210> 13 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> linker sequence <400> 13 ggtggttct 9 <210> 14 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> linker sequence <400> 14 ggatccggtg gc 12 <110> University-Industry Cooperation Foundation, Konkuk University <120> METHOD FOR PRODUCING ANTIMICROBIAL PEPTIDE USING INSOLUBLE GREEN FLUORESCENT PROTEIN AS A SCAFFOLD <130> P14-E610 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding Protegrin1 (PG1) <400> 1 aggggaggtc gcctgtgcta ttgtaggcgt aggttctgcg tctgtgtcgg acgagga 57 <210> 2 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding Pig myeloid antibacterial peptide36 (PMAP36) <400> 2 ggacgattta gacggttgcg taagaagacc cgaaaacgtt tgaagaagat cgggaaggtt 60 ttgaagtgga ttcctcccat tgtcggctca atacccttgg gttgtggg 108 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 catatgcatc accatcatca ccatcagagc 30 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 catggtacca gaaccacctt ccacgttatg ac 32 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 atgggatccg gtggcgatgg cagcgt 26 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 gtggtggtgc tcgagttatt aatggtg 27 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 gcggttctgg tggtaccatg aggggaggtc gcctgtg 37 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 gccaccggat cccattcctc gtccgacaca gacg 34 <210> 9 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 gcggttctgg tggtaccatg ggacgattta gacggttg 38 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 caccggatcc catcccacaa cccaagggta 30 <210> 11 <211> 843 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding recombinant protein of GFP and PG1 <400> 11 catatgcatc accatcatca ccatcagagc aaaggcgaag aactgtttac cggcgtggtg 60 ccgattctgg tggaactgga tggcgatgtg aacggccata aatttagcgt gcgtggcgaa 120 ggcgaaggcg atgcgaccaa cggcaaactg accctgaaat ttatttgcac caccggtaaa 180 ctgccggtgc cgtggccgac cctggtgacc accctgggtt atggtgtgca gtgctttgca 240 cgttatccgg atcacatcaa acgtcatgat ttctttaaaa gcgcgctgcc ggaaggctat 300 gtgcaggaac gtaccattag ctttaaagat gatggcacct ataaaacccg tgcggaagtg 360 aaatttgaag gcgataccct ggtgaaccgt attgaactga aaggcattga ttttaaagaa 420 gatggcaaca ttctgggcca taaactggaa tataacttta acagccataa agtgtatatt 480 accgcggata aacagaaaaa cggcattaaa gcgaacttta aaattcgtca taacgtggaa 540 ggtggttctg gtaccatgag gggaggtcgc ctgtgctatt gtaggcgtag gttctgcgtc 600 tgtgtcggac gaggaatggg atccggtggc gatggcagcg tgcagctggc ggatcattat 660 cagcagaaca ccccgattgg cgatgataac cattatctga gcacccagag cgtgctgctg 720 aaagatccga acgaaaaacg tgatcacgcg gtgctgctgg aatttgtgac cgcggcgggc 780 attacccacg gcaaagatga actgtataaa catcaccatc atcaccatta ataactcgag 840 atc 843 <210> 12 <211> 891 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding recombinant protein of GFP and PMAP36 <400> 12 catatgcatc accatcatca ccatcagagc aaaggcgaag aactgtttac cggcgtggtg 60 ccgattctgg tggaactgga tggcgatgtg aacggccata aatttagcgt gcgtggcgaa 120 ggcgaaggcg atgcgaccaa cggcaaactg accctgaaat ttatttgcac caccggtaaa 180 ctgccggtgc cgtggccgac cctggtgacc accctgggtt atggtgtgca gtgctttgca 240 cgttatccgg atcacatcaa acgtcatgat ttctttaaaa gcgcgctgcc ggaaggctat 300 gtgcaggaac gtaccattag ctttaaagat gatggcacct ataaaacccg tgcggaagtg 360 aaatttgaag gcgataccct ggtgaaccgt attgaactga aaggcattga ttttaaagaa 420 gatggcaaca ttctgggcca taaactggaa tataacttta acagccataa agtgtatatt 480 accgcggata aacagaaaaa cggcattaaa gcgaacttta aaattcgtca taacgtggaa 540 ggtggttctg gtaccatggg acgatttaga cggttgcgta agaagacccg aaaacgtttg 600 aagaagatcg ggaaggtttt gaagtggatt cctcccattg tcggctcaat acccttgggt 660 tgtgggatgg gatccggtgg cgatggcagc gtgcagctgg cggatcatta tcagcagaac 720 accccgattg gcgatgataa ccattatctg agcacccaga gcgtgctgct gaaagatccg 780 aacgaaaaac gtgatcacgc ggtgctgctg gaatttgtga ccgcggcggg cattacccac 840 ggcaaagatg aactgtataa acaccatcac caccatcact aataactcga g 891 <210> 13 <211> 9 <212> DNA <213> Artificial Sequence <220> Linker sequence <400> 13 ggtggttct 9 <210> 14 <211> 12 <212> DNA <213> Artificial Sequence <220> Linker sequence <400> 14 ggatccggtg gc 12
Claims (11)
A fusion protein in which an antibacterial peptide (AMP) is bound between amino acid sequences 172 and 173 of an insoluble green fluorescent protein (Green Fluorescent Protein, GFP) in which the methionine (Met) in the amino acid sequence is deleted.
A gene consisting of the nucleotide sequence of SEQ ID NO: 11.
A gene consisting of the nucleotide sequence of SEQ ID NO: 12.
An expression vector comprising the gene of claim 2.
An expression vector comprising the gene of claim 3.
An E. coli transformant transformed with the expression vector of claim 4.
An E. coli transformant transformed with the expression vector of claim 5.
A primer set for a polymerase chain reaction consisting of SEQ ID NO: 3 and SEQ ID NO: 4 for DNA amplification between 1 and 172 in the amino acid sequence of the insoluble green fluorescent protein in which the methionine (Met) in the amino acid sequence for producing an antimicrobial peptide is deleted .
A primer for a polymerase chain reaction consisting of SEQ ID NO: 5 and SEQ ID NO: 6 for DNA amplification between the 173rd to 3'end of the amino acid sequence of the insoluble green fluorescent protein in which the methionine (Met) was deleted in the amino acid sequence for the production of the antimicrobial peptide set.
(2) 상기 재조합된 유전자를 클로닝(cloning)하여 대장균 형질전환체를 제조하는 단계;
(3) 상기 제조된 대장균 형질전환체를 배양하여 재조합 단백질의 발현을 유도하는 단계;
(4) 상기 발현된 재조합 단백질을 분리 후 정제하는 단계; 및
(5) 상기 정제된 재조합 단백질에서 항균 펩타이드를 분리 후 정제하는 단계; 를 포함하는 항균 펩타이드의 제조방법.
(1) recombining a gene encoding an insoluble green fluorescent protein in which methionine (Met) in the amino acid sequence is deleted and a gene encoding an antimicrobial peptide;
(2) cloning the recombinant gene to prepare an E. coli transformant;
(3) culturing the E. coli transformant to induce expression of the recombinant protein;
(4) separating and purifying the expressed recombinant protein; And
(5) separating and purifying the antimicrobial peptide from the purified recombinant protein; ≪ / RTI >
상기 (1)단계에서 항균 펩타이드는 서열번호 1의 염기서열로 구성된 프로테그린1(Protegrin1, PG1) 또는 서열번호 2의 염기서열로 구성된 돼지 골수 유래 항균 펩타이드36(Pig myeloid antibacterial peptide36, PMAP36)인 것을 특징으로 하는 항균 펩타이드의 제조방법. 11. The method of claim 10,
In step (1), the antimicrobial peptide comprises Protegrin 1 (PG1) consisting of the nucleotide sequence of SEQ ID NO: 1 or Pig myeloid antibacterial peptide 36 (PMAP36) consisting of the nucleotide sequence of SEQ ID NO: 2 ≪ / RTI >
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CN108779152A (en) * | 2016-01-27 | 2018-11-09 | 建国大学校产学协力团 | Including the insoluble fusion protein of antibacterial peptide and the antibacterial peptide manufacturing method using above-mentioned insoluble fusion protein |
US11230575B2 (en) | 2016-01-27 | 2022-01-25 | Konkuk University Industrial Cooperation Corp | Insoluble fusion protein comprising antimicrobial peptide and method for producing antimicrobial peptide using same |
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