KR20210027132A - Method for producing monophosphoryl lipid A - Google Patents
Method for producing monophosphoryl lipid A Download PDFInfo
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- KR20210027132A KR20210027132A KR1020200107412A KR20200107412A KR20210027132A KR 20210027132 A KR20210027132 A KR 20210027132A KR 1020200107412 A KR1020200107412 A KR 1020200107412A KR 20200107412 A KR20200107412 A KR 20200107412A KR 20210027132 A KR20210027132 A KR 20210027132A
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- South Korea
- Prior art keywords
- lipid
- leu
- bacteria
- ala
- arg
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Abstract
Description
모노포스포릴 지질 A(monophosphoryl lipid A: MPLA)를 생산하는 세균으로부터 MPLA를 고순도 및 고효율로 생산하는 방법에 관한 것이다.It relates to a method for producing MPLA with high purity and high efficiency from bacteria producing monophosphoryl lipid A (MPLA).
지질다당류(lipopolysaccharide: LPS)는 그람-음성 세균의 펩티도글리칸을 둘러싸는 외막의 구성성분 중 하나이다. LPS는 지질 A(lipid A)와 이에 공유결합으로 결합하는 여러 종류의 당이 결합한 것으로, 그람-음성 세균의 독성을 담당하는 내독소(endotoxin)이다. LPS는 면역세포의 TLR4(Toll like receptor 4)에 작용하여 강력한 면역반응을 일으키지만, 매우 독성이 강하므로 지질 A만 분리하여 면역증강제로 사용하고 있다.Lipopolysaccharide (LPS) is one of the components of the outer membrane surrounding the peptidoglycan of Gram-negative bacteria. LPS is an endotoxin that is responsible for the toxicity of Gram-negative bacteria, which is a combination of lipid A and several types of sugars that covalently bind to it. LPS acts on TLR4 (Toll like receptor 4) of immune cells to cause a strong immune response, but because it is very toxic, only lipid A is isolated and used as an immunity enhancer.
지질 A는 두 개의 글루코사민(glucosamine) 골격에 1-,4'-인산기가 결합되어 있고, 4개의 아실 사슬은 글루코사민 머리 그룹의 2,3,2',3'에 직접적으로 결합되어 있다. 나머지 2개의 아실 사슬은 글루코사민 머리 그룹의 2'과 3'에 연결된 아실 사슬의 히드록시기에 결합되어 있다. LPS가 내독소로 작용하는 기전에서 글루코사민 골격의 1번 탄소에 결합된 인산기가 중요한 역할을 하게 된다. MPLA(또는 1-탈인산 지질 A)는 지질 A의 구조에서 1번 글루코사민 머리 그룹의 인산기가 히드록시기로 치환된 것으로, 부작용이 심한 지질 A와 달리, 부작용은 없으면서 면역증강 효능이 우수하다.Lipid A has a 1-,4'-phosphate group attached to two glucosamine backbones, and four acyl chains are directly attached to 2,3,2',3' of the glucosamine head group. The other two acyl chains are bound to the hydroxy groups of the acyl chain linked to the 2'and 3'of the glucosamine head group. In the mechanism by which LPS acts as an endotoxin, the phosphate group bonded to
최근, 헥사 아실화된 MPLA를 생산하는 세균 및 이를 이용한 헥사 아실화된 모노포스포릴 지질 A 생산 방법에 대해 개발된 바 있다(한국 특허번호 10-1761348(2017.07.19)).Recently, a bacterium producing hexaacylated MPLA and a method for producing hexaacylated monophosphoryl lipid A using the same have been developed (Korean Patent No. 10-1761348 (2017.07.19)).
그러나, 세균의 외막에는 지질 A와 MPLA가 함께 함유되어 있고 이들이 구조적으로 매우 유사하기 때문에, 두 물질을 분리하여 MPLA를 고순도 및 고효율로 생산하는 방법을 개발할 필요가 있다.However, since the outer membrane of bacteria contains lipid A and MPLA together and they are structurally very similar, there is a need to develop a method for producing MPLA with high purity and high efficiency by separating the two substances.
모노포스포릴 지질 A를 고순도 또는 고효율로 생산하는 방법을 제공한다.It provides a method of producing monophosphoryl lipid A with high purity or high efficiency.
일 양상은 모노포스포릴 지질 A를 생산하는 세균을 배양하는 단계로서, 상기 세균은 생장곡선에서 지수기(exponential phase) 시작점으로부터 정지기(stationary phase) 시작점 후 5시간까지 배양되는 것인 단계; 배양물로부터 상기 세균을 수득하는 단계; 수득된 세균으로부터 지질을 수득하는 단계; 및 수득된 지질로부터 모노포스포릴 지질 A를 수득하는 단계를 포함하는 모노포스포릴 지질 A(monophosphoryl lipid A: MPLA)를 생산하는 방법을 제공한다.One aspect is the step of culturing a bacterium producing monophosphoryl lipid A, wherein the bacterium is cultured from the starting point of the exponential phase to the starting point of the stationary phase in the growth curve for up to 5 hours; Obtaining the bacteria from the culture; Obtaining lipids from the obtained bacteria; And it provides a method for producing monophosphoryl lipid A (monophosphoryl lipid A: MPLA) comprising the step of obtaining monophosphoryl lipid A from the obtained lipid.
모노포스포릴 지질 A의 지질 A는 2개의 글루코사민(탄수화물 또는 당)에 부착된 아실 사슬로 이루어져 있고, 일반적으로 각 글루코사민에 하나의 인산기를 함유한다. 아실 사슬의 개수에 따라 트리, 테트라, 펜타, 헥사, 또는 헵타 아실화된 지질 A일 수 있다. 글루코사민에 직접 부착된 4개의 아실 사슬은 길이가 10 내지 16개의 탄소로 이루어진 베타 히드록시 아실 사슬이고, 2개의 추가적인 아실 사슬은 주로 베타 히드록시기에 부착된 것일 수 있다. Lipid A of monophosphoryl lipid A consists of an acyl chain attached to two glucosamines (carbohydrates or sugars), and generally contains one phosphate group in each glucosamine. Depending on the number of acyl chains, it may be tri, tetra, penta, hexa, or hepta acylated lipid A. The four acyl chains directly attached to glucosamine are beta hydroxy acyl chains consisting of 10 to 16 carbons in length, and the two additional acyl chains may be mainly attached to a beta hydroxy group.
모노포스포릴 지질 A(monophosphoryl lipid A: MPLA)는 2개의 글루코사민 중 하나에만 1개의 인산기가 결합된 모노포스포릴 지질 A를 말한다. 상기 MPLA는 트리, 테트라, 펜타, 헥사, 또는 헵타 아실화된 MLA일 수 있다. 예를 들어, 상기 MPLA는 1-탈인산-지질 A, 1-탈인산-펜타 아실 지질 A, 1-탈인산-테트라 아실 지질 A, 또는 이들의 조합일 수 있다.Monophosphoryl lipid A (MPLA) refers to monophosphoryl lipid A in which one phosphate group is bound to only one of two glucosamines. The MPLA may be tri, tetra, penta, hexa, or hepta acylated MLA. For example, the MPLA may be 1-dephosphoric acid-lipid A, 1-dephosphoric acid-pentaacyl lipid A, 1-dephosphoric acid-tetraacyl lipid A, or a combination thereof.
상기 MPLA는 당 모이어티(sugar moiety)를 포함하지 않는 것일 수 있다. 상기 당 모이어티는 2-케토-3-데옥시-D-만노-옥투로소네이트(2-keto-3-deoxy-D-manno-octulosonate: Kdo)일 수 있다. Kdo는 지질다당류(LPS)의 구성 성분으로서, 거의 모든 LPS에서 발견되는 보존된 잔기이다.The MPLA may not contain a sugar moiety. The sugar moiety may be 2-keto-3-deoxy-D-manno-octulosonate (Kdo). Kdo is a constituent of lipopolysaccharide (LPS) and is a conserved residue found in almost all LPS.
상기 MPLA는 살아있는 세균의 막, 예를 들어 외막에 존재하는 것일 수 있다.The MPLA may be present in the membrane of living bacteria, for example, the outer membrane.
상기 방법은 모노포스포릴 지질 A를 생산하는 세균을 배양하는 단계를 포함한다.The method includes culturing a bacterium that produces monophosphoryl lipid A.
용어 "세균(bacteria)"는 원핵 세균을 말한다. 상기 세균은 그람-음성(Gram-negative) 세균일 수 있다. 그람-음성 세균은 그람 염색법에 사용되는 크리스탈 바이올렛으로 염색되지 않는 종류의 세균을 말한다. 그람-음성 세균의 막은 각각의 이중막으로 이루어진 외막과 내막으로 이루어져 있고, 얇은 펩티도글리칸 층이 존재한다. 상기 세균은 에스케리치아(Escherichia) 속 세균, 시겔라(Shigella) 속 세균, 살모넬라(Salmonella) 속 세균, 캄피로박터(Campylobacter) 속 세균, 네이세리아(Neisseria) 속 세균, 헤모필루스(Haemophilus) 속 세균, 아에로모나스(Aeromonas) 속 세균, 프란시셀라(Francisella) 속 세균, 예르시니아(Yersinia) 속 세균, 클레브시엘라(Klebsiella) 속 세균, 보르데텔라(Bordetella) 속 세균, 레지오넬라(Legionella) 속 세균, 코리네박테리아(Corynebacteria) 속 세균, 시트로박터(Citrobacter) 속 세균, 클라미디아(Chlamydia) 속 세균, 브루셀라(Brucella) 속 세균, 슈도모나스(Pseudomonas) 속 세균, 헬리코박터(Helicobacter) 속 세균, 부르크홀데리아(Burkholderia) 속 세균, 포르피로모나스(Porphyromonas) 속 세균, 리조비움(Rhizobium) 속 세균, 메소리조비움(Mesorhizobium) 속 세균 및 비브리오(Vibrio) 속 세균으로 이루어진 군으로부터 선택된 세균일 수 있다. 세균은 예를 들어 대장균이다.The term "bacteria" refers to prokaryotic bacteria. The bacteria may be Gram-negative bacteria. Gram-negative bacteria are types of bacteria that are not stained with crystal violet, which is used in the Gram staining method. The membrane of Gram-negative bacteria consists of an outer membrane and an inner membrane of each double membrane, and a thin layer of peptidoglycan exists. The bacteria are Escherichia genus bacteria, Shigella genus bacteria, Salmonella genus bacteria, Campylobacter genus bacteria, Neisseria genus bacteria, Haemophilus genus bacteria , Aeromonas genus bacteria, Francisella genus bacteria, Yersinia genus bacteria, Klebsiella genus bacteria, Bordetella genus bacteria, Legionella Legionella), Corynebacteria, Citrobacter, Chlamydia, Brucella, Pseudomonas, Pseudomonas, Helicobacter , Burkholderia genus, Porphyromonas genus bacteria, Rhizobium genus bacteria, Mesorhizobium genus bacteria, and Vibrio genus bacteria may be selected from the group consisting of bacteria. have. The bacterium is, for example, E. coli.
상기 세균은 발현 유도제(예, 이소프로필 β-D-1-티오갈락토피라노시드(Isopropyl β-D-1-thiogalactopyranoside: IPTG)) 또는 발현 유도 자극(예, 열처리)의 존재 유무와 관계없이 MPLA를 생산할 수 있다.The bacteria are irrespective of the presence or absence of an expression inducing agent (e.g., isopropyl β-D-1-thiogalactopyranoside (IPTG)) or an expression inducing stimulus (e.g., heat treatment). Can produce MPLA.
상기 세균은 지질 A-1 포스파타아제(Lipid A-1 phosphatase: LpxE) 폴리펩티드, 지질 A 생합성 라우로일전이효소(lipid A biosysthesis lauroyltransferase: LpxL) 폴리펩티드, 지질 A 생합성 미리스토일전이효소(lipid A biosysthesis myristoyltransferase: LpxM) 폴리펩티드, P또는 이들의 조합을 포함할 수 있다.The bacterium is a lipid A-1 phosphatase (LpxE) polypeptide, a lipid A biosysthesis lauroyltransferase (LpxL) polypeptide, a lipid A biosynthetic myristoyl transferase (lipid A). biosysthesis myristoyltransferase: LpxM) polypeptide, P, or a combination thereof.
상기 LpxE 폴리펩티드는 3개 아미노산으로 이루어진(tripartite) 활성 부위와 6 개의 막 통과 헬릭스를 포함하는 지질 인산 포스파타아제에 속한다. 지질 인산 포스파타아제는 인산 모노에스테르 결합에 특이적으로 결합하는 가수분해효소로서, 인산기를 포함하는 지질로부터 인산기(phosphate group)를 제거할 수 있다. 상기 LpxE 폴리펩티드는 헬리코박터(Helicobacter) 속 세균(예, Helicobactor pylori), 아퀴펙스(Aquifex) 속 세균, 프란시셀라(Francisella) 속 세균, 보르데텔라(Bordetella) 속 세균, 브루셀라(Brucella) 속 세균, 리조비움(Rhizobium) 속 세균, 메소리조비움(Mesorhizobium) 속 세균, 및 포르피로모나스(Porphyromonas) 속 세균으로 이루어지는 군으로부터 선택된 세균의 LpxE 폴리펩티드일 수 있다. 상기 LpxE 폴리펩티드는 서열번호 12의 아미노산 서열, 또는 서열번호 12의 아미노산 서열과 약 90% 동일성, 약 80% 동일성, 약 70% 동일성, 약 60% 동일성, 약 50% 동일성, 약 40% 동일성, 약 30% 동일성, 약 20% 동일성, 또는 약 10% 동일성을 갖는 아미노산 서열을 포함하는 폴리펩티드일 수 있다. 상기 LpxE 폴리펩티드는 서열번호 13의 핵산 서열, 또는 서열번호 10의 핵산 서열과 90% 동일성, 80% 동일성, 70% 동일성, 60% 동일성, 50% 동일성, 40% 동일성, 30% 동일성, 20% 동일성, 또는 10% 동일성을 갖는 핵산 서열을 포함하는 폴리뉴클레오티드에 의해 코딩되는 것일 수 있다. 상기 LpxE 폴리펩티드는 돌연변이된 것일 수 있다. 상기 폴리뉴클레오티드는 예를 들어 야행형 LpxE 폴리펩티드의 N-말단으로부터 17번째 아미노산인 세린(17Ser)을 코딩하는 뉴클레오티드 서열을 AGC에서 TCG로 돌연변이시킨 것이다.The LpxE polypeptide belongs to a lipid phosphate phosphatase comprising an active site consisting of three amino acids (tripartite) and six transmembrane helixes. Lipid phosphate phosphatase is a hydrolase that specifically binds to a phosphate monoester bond, and can remove a phosphate group from a lipid containing a phosphate group. The LpxE polypeptide is a bacterium of the genus Helicobacter (eg, Helicobactor pylori), a bacterium of the genus Aquifex, a bacterium of the genus Francisella, a bacterium of the genus Bordetella, a bacterium of the genus Brucella, It may be an LpxE polypeptide of a bacterium selected from the group consisting of a bacterium of the genus Rhizobium, a bacterium of the genus Mesorhizobium, and a bacterium of the genus Porphyromonas. The LpxE polypeptide is about 90% identity, about 80% identity, about 70% identity, about 60% identity, about 50% identity, about 40% identity, about the amino acid sequence of SEQ ID NO: 12, or the amino acid sequence of SEQ ID NO: 12. It may be a polypeptide comprising an amino acid sequence having 30% identity, about 20% identity, or about 10% identity. The LpxE polypeptide has 90% identity, 80% identity, 70% identity, 60% identity, 50% identity, 40% identity, 30% identity, 20% identity with the nucleic acid sequence of SEQ ID NO: 13 or the nucleic acid sequence of SEQ ID NO: 10 , Or may be encoded by a polynucleotide comprising a nucleic acid sequence having 10% identity. The LpxE polypeptide may be mutated. The polynucleotide is, for example, a nucleotide sequence encoding serine (17Ser), which is the 17th amino acid from the N-terminus of the nocturnal LpxE polypeptide, is mutated from AGC to TCG.
상기 LpxL 폴리펩티드는 지질 A 생합성 라우로일전이효소(lauroyltransferase)로서, 라우로일-아실 전달 단백질(acyl carrier protein: ACP)로부터 Kdo2-지질 IVA로 라우레이트(laurate)를 전이하여 Kdo2-(라우로일)-지질 IVA를 형성하는 것을 촉매하는 효소이다. 상기 LpxL 폴리펩티드는 에스케리치아 속 세균, 시겔라 속 세균, 살모넬라 속 세균, 캄피로박터 속 세균, 네이세리아 속 세균, 헤모필루스 속 세균, 아에로모나스 속 세균, 프란시셀라 속 세균, 예르시니아 속 세균, 클레브시엘라 속 세균, 보르데텔라 속 세균, 레지오넬라 속 세균, 코리네박테리아속, 시트로박터 속 세균, 클라미디아 속 세균, 브루셀라 속 세균, 슈도모나스 속 세균, 헬리코박터 속 세균, 부르크홀데리아 속 세균, 포르피토모나스 속 세균, 리조비움 속 세균, 메소리조비움 속 세균, 및 비브리오 속 세균으로 이루어진 군으로부터 선택된 세균의 LpxL 폴리펩티드일 수 있다. 예를 들어, 상기 LpxL 폴리펩티드는 대장균의 LpxL 폴리펩티드(EcLpxL)일 수 있다. 상기 LpxL 폴리펩티드는 서열번호 1의 아미노산 서열, 또는 서열번호 1의 아미노산 서열과 약 90% 동일성, 약 80% 동일성, 약 70% 동일성, 약 60% 동일성, 약 50% 동일성, 약 40% 동일성, 약 30% 동일성, 약 20% 동일성, 또는 약 10% 동일성을 갖는 아미노산 서열을 포함하는 폴리펩티드일 수 있다. 상기 LpxL 폴리펩티드는 서열번호 2의 핵산 서열, 또는 서열번호 2의 핵산 서열과 90% 동일성, 80% 동일성, 70% 동일성, 60% 동일성, 50% 동일성, 40% 동일성, 30% 동일성, 20% 동일성, 또는 10% 동일성을 갖는 핵산 서열을 포함하는 폴리뉴클레오티드에 의해 코딩되는 것일 수 있다.The LpxL polypeptide is a lipid A biosynthetic lauroyltransferase, which transfers laurate from lauroyl-acyl carrier protein (ACP) to Kdo 2 -lipid IV A to Kdo 2- (Lauroyl)-is an enzyme that catalyzes the formation of lipid IV A. The LpxL polypeptide is a bacterium of the genus Escherichia, a bacterium of the genus Shigella, a bacterium of the genus Salmonella, a bacterium of the genus Campyrobacter, a bacterium of the genus Neisseria, a bacterium of the genus Haemophilus, a bacterium of the genus Aeromonas, a bacterium of the genus Francis Genus bacteria, Klebsiella genus, Bordetella genus, Legionella genus, Corynebacteria genus, Citrobacter genus, Chlamydia genus, Brucella genus, Pseudomonas genus, Helicobacter genus, Burgholderia It may be an LpxL polypeptide of a bacterium selected from the group consisting of genus bacteria, Porphytomonas genus, Rhizobium genus, Mesozobium genus bacteria, and Vibrio genus bacteria. For example, the LpxL polypeptide may be an E. coli LpxL polypeptide (EcLpxL). The LpxL polypeptide is about 90% identity, about 80% identity, about 70% identity, about 60% identity, about 50% identity, about 40% identity, about the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 It may be a polypeptide comprising an amino acid sequence having 30% identity, about 20% identity, or about 10% identity. The LpxL polypeptide has 90% identity, 80% identity, 70% identity, 60% identity, 50% identity, 40% identity, 30% identity, 20% identity with the nucleic acid sequence of SEQ ID NO: 2 or the nucleic acid sequence of SEQ ID NO: 2 , Or may be encoded by a polynucleotide comprising a nucleic acid sequence having 10% identity.
상기 LpxM 폴리펩티드는 지질 A 생합성 미리스토일전이효소(myristoyltransferase)로서, 미리스토일-아실 전달 단백질로부터 Kdo2-라우로일-지질 IVA로 미리스테이트(myristate)를 전이하여 Kdo2-지질 A를 형성하는 것을 촉매하는 효소이다. 상기 LpxM 폴리펩티드는 에스케리치아 속 세균, 시겔라 속 세균, 살모넬라 속 세균, 캄피로박터 속 세균, 네이세리아 속 세균, 헤모필루스 속 세균, 아에로모나스 속 세균, 프란시셀라 속 세균, 예르시니아 속 세균, 클레브시엘라 속 세균, 보르데텔라 속 세균, 레지오넬라 속 세균, 코리네박테리아속, 시트로박터 속 세균, 클라미디아 속 세균, 브루셀라 속 세균, 슈도모나스 속 세균, 헬리코박터 속 세균, 부르크홀데리아 속 세균, 포르피토모나스 속 세균, 리조비움 속 세균, 메소리조비움 속 세균, 및 비브리오 속 세균으로 이루어진 군으로부터 선택된 세균의 LpxM 폴리펩티드일 수 있다. 예를 들어, 상기 LpxM 폴리펩티드는 대장균의 LpxM 폴리펩티드(EcLpxM)일 수 있다. 상기 LpxM 폴리펩티드는 서열번호 5의 아미노산 서열, 또는 서열번호 5의 아미노산 서열과 약 90% 동일성, 약 80% 동일성, 약 70% 동일성, 약 60% 동일성, 약 50% 동일성, 약 40% 동일성, 약 30% 동일성, 약 20% 동일성, 또는 약 10% 동일성을 갖는 아미노산 서열을 포함하는 폴리펩티드일 수 있다. 상기 LpxM 폴리펩티드는 서열번호 6의 핵산 서열, 또는 서열번호 6의 핵산 서열과 90% 동일성, 80% 동일성, 70% 동일성, 60% 동일성, 50% 동일성, 40% 동일성, 30% 동일성, 20% 동일성, 또는 10% 동일성을 갖는 핵산 서열을 포함하는 폴리뉴클레오티드에 의해 코딩되는 것일 수 있다.The LpxM polypeptide is a lipid A biosynthetic myristoyltransferase, which transfers myristate from myristoyl-acyl transfer protein to Kdo 2 -lauroyl-lipid IV A to transfer Kdo 2 -lipid A. It is an enzyme that catalyzes formation. The LpxM polypeptide is a bacterium of the genus Escherichia, a bacterium of the genus Shigella, a bacterium of the genus Salmonella, a bacterium of the genus Campyrobacter, a bacterium of the genus Neisseria, a bacterium of the genus Haemophilus, a bacterium of the genus Aeromonas, a bacterium of the genus Francis Genus bacteria, Klebsiella genus, Bordetella genus, Legionella genus, Corynebacteria genus, Citrobacter genus, Chlamydia genus, Brucella genus, Pseudomonas genus, Helicobacter genus, Burgholderia It may be an LpxM polypeptide of a bacterium selected from the group consisting of genus bacteria, Porphytomonas genus, Rhizobium genus, Mesozobium genus bacteria, and Vibrio genus bacteria. For example, the LpxM polypeptide may be an E. coli LpxM polypeptide (EcLpxM). The LpxM polypeptide is about 90% identity, about 80% identity, about 70% identity, about 60% identity, about 50% identity, about 40% identity, about the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 5 It may be a polypeptide comprising an amino acid sequence having 30% identity, about 20% identity, or about 10% identity. The LpxM polypeptide has 90% identity, 80% identity, 70% identity, 60% identity, 50% identity, 40% identity, 30% identity, 20% identity with the nucleic acid sequence of SEQ ID NO: 6 or the nucleic acid sequence of SEQ ID NO: 6 , Or may be encoded by a polynucleotide comprising a nucleic acid sequence having 10% identity.
상기 세균은 LpxT 폴리펩티드를 코딩하는 폴리뉴클레오티드, PagP 폴리펩티드를 코딩하는 폴리뉴클레오티드, KdtA 폴리펩티드를 코딩하는 폴리뉴클레오티드, 또는 이들의 조합이 돌연변이된 것일 수 있다. 상기 LpxT 폴리펩티드는 내막 단백질(Inner membrane protein) LpxT이다. 상기 PagP 폴리펩티드는 지질 A 팔미토일전이효소(palmitoyltransferase)로서, 팔미테이트(palmitate)를 함유하는 헵타-아실 지질 A 종류의 생합성에 필요한 폴리펩티드이다. 상기 KdtA 폴리펩티드는 지질 IVA에 Kdo를 부착시키는 효소일 수 있다. 상기 돌연변이(mutation)는 유전물질의 변이를 말하고, 점 돌연변이, 프레임 시프트(frame shift) 돌연변이, 삽입, 결실, 역위, 전좌 등을 포함한다. 상기 돌연변이에 의해 상기 세균의 게놈으로부터 결실 또는 도입될 수 있다.The bacterium may be a polynucleotide encoding an LpxT polypeptide, a polynucleotide encoding a PagP polypeptide, a polynucleotide encoding a KdtA polypeptide, or a combination thereof mutated. The LpxT polypeptide is an inner membrane protein LpxT. The PagP polypeptide is a lipid A palmitoyltransferase, which is a polypeptide necessary for biosynthesis of hepta-acyl lipid A type containing palmitate. The KdtA polypeptide may be an enzyme that attaches Kdo to lipid IV A. The mutation refers to a mutation in genetic material, and includes point mutation, frame shift mutation, insertion, deletion, inversion, translocation, and the like. It can be deleted or introduced from the genome of the bacterium by the mutation.
상기 세균은 그의 세균 염색체에서 운데카프레닐 피로포스페이트 포스파타제(undecaprenyl pyrophosphate phosphatase: Und-PP 포스파타제)를 암호화하는 폴리뉴클레오티드, 포스파티딜글리세로포스페이트 포스파타제(phosphatidylglycerophosphate phosphatase: PGP 포스파타제)를 암호화하는 폴리뉴클레오티드, 또는 이들의 조합이 돌연변이된 것일 수 있다. 용어 "세균 염색체(bacterial chromosome)"는 세균의 유전 정보로서, 환형의 DNA일 수 있다. 상기 세균 염색체는 플라스미드를 제외할 수 있다. 플라스미드(plasmid)는 세균 염색체와 물리적으로 분리되어 있고 독립적으로 복제할 수 있는 환형의 DNA를 말한다.The bacterium is a polynucleotide encoding undecaprenyl pyrophosphate phosphatase (Und-PP phosphatase) in its bacterial chromosome, a polynucleotide encoding phosphatidylglycerophosphate phosphatase (PGP phosphatase), or these The combination of may be mutated. The term "bacterial chromosome" is genetic information of a bacterium, and may be circular DNA. The bacterial chromosome may exclude a plasmid. Plasmid refers to a circular DNA that is physically separated from a bacterial chromosome and can be replicated independently.
상기 Und-PP 포스파타제는 운데카프레닐 피로포스페이트의 탈인산화를 촉매하여 운데카프레닐 포스페이트를 생산하는 효소이다. 운데카프레닐 포스페이트는 세균의 외피(envelope)로 보내지는 탄화수소 중합체를 위한 글리칸 생합성 중간체(intermediate)의 지질 운반체이다. 상기 Und-PP 포스파타제를 암호화하는 폴리뉴클레오티드는 bacA 유전자, pgpB 유전자, ybjG 유전자, 또는 이들의 조합일 수 있다. bacA 유전자는 과발현시 항생물질인 바시트라신(bacitracin)에 저항성을 야기하는 유전자이다. pgpB 유전자는 파스파티딜글리세롤 포스페이트(phosphatidylglycerol phosphate: PGP)를 탈인산화시켜 포스파티딜 글리세롤(Phosphatidyl glycerol: PG)을 생성하는 과정을 촉매하는 효소를 암호화하는 유전자로서, 상기 효소는 Und-PP 포스파타제 활성을 갖는 것일 수 있다. 상기 ybjG 유전자는 과발현시 Und-PP 포스파타제 활성을 증가시키고 바시트라신에 대한 저항성을 증가시킬 수 있는 유전자이다.The Und-PP phosphatase is an enzyme that catalyzes the dephosphorylation of undecaprenyl pyrophosphate to produce undecaprenyl phosphate. Undecaprenyl phosphate is a lipid carrier of the glycan biosynthetic intermediate for hydrocarbon polymers that are sent to the bacterial envelope. The polynucleotide encoding the Und-PP phosphatase may be a bacA gene, a pgpB gene, a ybjG gene, or a combination thereof. The bacA gene is a gene that causes resistance to the antibiotic bacitracin when overexpressed. The pgpB gene is a gene encoding an enzyme that catalyzes the process of dephosphorylating phosphatidylglycerol phosphate (PGP) to produce phosphatidyl glycerol (PG), and the enzyme has Und-PP phosphatase activity. Can be. The ybjG gene is a gene capable of increasing Und-PP phosphatase activity and resistance to bacitracin upon overexpression.
상기 PGP 포스파타제를 암호화하는 폴리뉴클레오티드는 pgpB 유전자, pgpA 유전자, pgpC 유전자, 또는 이들의 조합일 수 있다. pgpA 유전자 또는 pgpC 유전자는 PGP를 포스파티딜글리세롤로 탈인산화시키는 지질 포스파타제를 암호화하는 유전자이다.The polynucleotide encoding the PGP phosphatase may be a pgpB gene, a pgpA gene, a pgpC gene, or a combination thereof. The pgpA gene or the pgpC gene is a gene encoding a lipid phosphatase that dephosphorylates PGP to phosphatidylglycerol.
용어 "유전자(gene)"은 유전 정보의 단위로서, 폴리펩티드를 암호화하는 열린 해독 틀(open reading frame: ORF) 및 유전자의 전사(transcription)를 조절하는 조절 서열(regulatory sequence)을 포함할 수 있다. 상기 조절 서열은 유전자의 전사를 개시하는 DNA 영역인 프로모터(promoter), 전사를 촉진할 수 있는 인핸서(enhancer), 전사를 억제할 수 있는 사일런서(silencer), 또는 이들의 조합을 포함할 수 있다.The term "gene" is a unit of genetic information and may include an open reading frame (ORF) encoding a polypeptide and a regulatory sequence that regulates transcription of a gene. The regulatory sequence may include a promoter, which is a DNA region that initiates transcription of a gene, an enhancer capable of promoting transcription, a silencer capable of inhibiting transcription, or a combination thereof.
용어 "돌연변이(mutation)"는 유전 물질의 변이를 말하고, 점 돌연변이, 격자 이동(frame shift) 돌연변이, 삽입, 결실, 역위, 전좌 등을 포함한다. 돌연변이는 결실(deletion), 삽입(insertion), 점 돌연변이(point mutation), 격자 이동 돌연변이(frame shift mutation), 또는 이들의 조합일 수 있다. 상기 점 돌연변이는 미스센스(missense) 돌연변이 또는 넌센스(nonsense) 돌연변이일 수 있다. 상기 돌연변이에 의해 유전 물질이 상기 세균의 게놈으로부터 결실 또는 도입될 수 있다.The term “mutation” refers to mutations in genetic material and includes point mutations, frame shift mutations, insertions, deletions, inversions, translocations, and the like. The mutation may be deletion, insertion, point mutation, frame shift mutation, or a combination thereof. The point mutation may be a missense mutation or a nonsense mutation. Genetic material may be deleted or introduced from the bacterial genome by the mutation.
배양액의 종류, 배양 온도, 배양 조건 등은 공지된 것일 수 있다. 배양 배지는 항생제를 포함할 수 있다. 상기 항생제는 예를 들어 카나마이신, 암피실린, 클로람피니콜 또는 이들의 조합이다.The type of culture medium, culture temperature, culture conditions, etc. may be known. The culture medium may contain antibiotics. The antibiotic is, for example, kanamycin, ampicillin, chlorampinicol or a combination thereof.
상기 세균은 생장곡선에서 지수기(exponential phase) 시작점으로부터 정지기(stationary phase) 시작점 후 약 5시간까지 배양된 것일 수 있다. 세균의 생장곡선은 지체기(lag phase), 지수기(exponential phase 또는 log phase), 정지기(stationary phase), 및 사멸기(death phase)로 구분될 수 있다. 지체기는 세균이 성장 조건에 적응하여 세균이 아직 분열할 수 없는 기간을 말한다. 지수기는 세균이, 성장이 제한되지 않는 한, 지수적으로 분열하는 기간을 말한다. 정지기는 중요한 영양성분의 고갈 또는 유기산과 같은 억제 생성물의 형성 등에 의해 시작되며 세균 사멸 속도와 세균의 증식 속도가 같은 기간을 말한다. 사멸기는 영양성분의 고갈 등으로 세균의 증식 속도가 감소하는 기간을 말한다. 상기 세균은 지수기의 시작 시점으로부터 정지기의 시작 시점, 정지기의 시작 시점 후 약 1시간, 정지기의 시작 시점 후 약 1시간 30분, 정지기의 시작 시점 후 약 2시간, 정지기의 시작 시점 후 약 2시간 30분, 정지기의 시작 시점 후 약 3시간, 정지기의 시작 시점 후 약 3시간 30분, 정지기의 시작 시점 후 약 4시간, 정지기의 시작 시점 후 약 4시간 30분, 정지기의 시작 시점 후 약 5시간 미만, 또는 정지기의 시작 시점 후 약 5시간까지의 시간 동안 배양된 것일 수 있다.The bacteria may be cultured from the start point of the exponential phase to about 5 hours after the start point of the stationary phase in the growth curve. The growth curve of bacteria can be divided into a lag phase, an exponential phase or a log phase, a stationary phase, and a death phase. The retardation period refers to the period during which the bacteria cannot yet divide by adapting to the growing conditions. The exponential phase refers to the period during which bacteria divide exponentially, unless growth is limited. The stationary phase is initiated by the depletion of important nutrients or the formation of inhibitory products such as organic acids, and refers to a period in which the rate of bacterial death and the rate of bacterial growth are the same. Death period refers to the period during which the growth rate of bacteria decreases due to depletion of nutrients. The bacteria are from the start of the exponential phase to the start of the stationary phase, about 1 hour after the start of the stationary phase, about 1 hour and 30 minutes after the start of the stationary phase, about 2 hours after the start of the stationary phase, and About 2 hours and 30 minutes after the start, about 3 hours after the start of the stop, about 3 hours and 30 minutes after the start of the stop, about 4 hours after the start of the stop, about 4 hours after the start of the stop It may be cultured for 30 minutes, less than about 5 hours after the start of the stationary phase, or up to about 5 hours after the start of the stoppage.
상기 세균은 파장 600 nm에서의 흡광도(optical density(OD) 또는 absorbance)가 약 10 내지 약 70, 약 15 내지 약 70, 약 20 내지 약 70, 약 25 내지 약 70, 약 30 내지 약 70, 약 35 내지 약 70, 약 40 내지 약 70, 약 45 내지 약 70, 약 50 내지 약 70, 약 55 내지 약 70, 약 50 내지 약 68, 약 50 내지 약 66, 약 50 내지 약 64, 약 50 내지 약 62, 약 50 내지 약 61, 또는 약 55 내지 약 62의 범위까지 배양할 수 있다.The bacterium has an optical density (OD) or absorbance at a wavelength of 600 nm of about 10 to about 70, about 15 to about 70, about 20 to about 70, about 25 to about 70, about 30 to about 70, about 35 to about 70, about 40 to about 70, about 45 to about 70, about 50 to about 70, about 55 to about 70, about 50 to about 68, about 50 to about 66, about 50 to about 64, about 50 to It can be cultured to a range of about 62, about 50 to about 61, or about 55 to about 62.
상기 세균은 약 10 시간 내지 약 30 시간, 약 12 시간 내지 약 30 시간, 약 14 시간 내지 약 30 시간, 약 16 시간 내지 약 30 시간, 약 18 시간 내지 약 30 시간, 약 20 시간 내지 약 30 시간, 약 21 시간 내지 약 30 시간, 약 21 시간 내지 약 29 시간, 약 21 시간 내지 약 28 시간, 약 21 시간 내지 약 27 시간, 약 21 시간 내지 약 26 시간, 약 21 시간 내지 약 25 시간, 약 21 시간 내지 약 24 시간, 약 21 시간 내지 약 23 시간, 또는 약 21 시간 내지 약 22 시간 동안 배양될 수 있다.The bacterium is about 10 hours to about 30 hours, about 12 hours to about 30 hours, about 14 hours to about 30 hours, about 16 hours to about 30 hours, about 18 hours to about 30 hours, about 20 hours to about 30 hours , About 21 hours to about 30 hours, about 21 hours to about 29 hours, about 21 hours to about 28 hours, about 21 hours to about 27 hours, about 21 hours to about 26 hours, about 21 hours to about 25 hours, about 21 hours to about 24 hours, about 21 hours to about 23 hours, or about 21 hours to about 22 hours.
상기 세균은 약 25℃ 내지 약 40℃, 약 26℃ 내지 약 40℃, 약 27℃ 내지 약 40℃, 약 28℃ 내지 약 40℃, 약 29℃ 내지 약 40℃, 약 30℃ 내지 약 40℃, 약 31℃ 내지 약 40℃, 약 32℃ 내지 약 40℃, 약 33℃ 내지 약 40℃, 약 34℃ 내지 약 40℃, 약 35℃ 내지 약 40℃, 약 36℃ 내지 약 40℃, 약 37℃ 내지 약 40℃, 약 30℃ 내지 약 39℃, 약 30℃ 내지 약 38℃, 약 30℃ 내지 약 37℃, 약 30℃ 내지 약 36℃, 약 30℃ 내지 약 35℃, 약 30℃ 내지 약 34℃, 약 30℃ 내지 약 33℃, 약 30℃ 내지 약 32℃, 또는 약 30℃ 내지 약 31℃의 온도에서 배양될 수 있다.The bacteria are about 25 ℃ to about 40 ℃, about 26 ℃ to about 40 ℃, about 27 ℃ to about 40 ℃, about 28 ℃ to about 40 ℃, about 29 ℃ to about 40 ℃, about 30 ℃ to about 40 ℃ , About 31°C to about 40°C, about 32°C to about 40°C, about 33°C to about 40°C, about 34°C to about 40°C, about 35°C to about 40°C, about 36°C to about 40°C, about 37 ℃ to about 40 ℃, about 30 ℃ to about 39 ℃, about 30 ℃ to about 38 ℃, about 30 ℃ to about 37 ℃, about 30 ℃ to about 36 ℃, about 30 ℃ to about 35 ℃, about 30 ℃ It can be cultured at a temperature of about 34°C, about 30°C to about 33°C, about 30°C to about 32°C, or about 30°C to about 31°C.
상기 세균의 배양 방법은 공지된 것일 수 있다. 배양 방법은 예를 들어, 회분 배양(batch culture), 유가 배양(fed-batch culture), 연속 배양(continuous culture), 발효(fermentation), 또는 이들의 조합이다. 세균은 배양시 진탕하면서 배양될 수 있다. The method of culturing the bacteria may be known. The culture method is, for example, batch culture, fed-batch culture, continuous culture, fermentation, or a combination thereof. Bacteria can be cultured while shaking during culture.
상기 방법은 배양물로부터 상기 세균을 수득하는 단계를 포함한다.The method includes obtaining the bacterium from the culture.
배양물로부터 세균을 수득하는 방법은 공지된 것일 수 있다. 예를 들어 원심분리, 여과, 또는 이들의 조합을 통해 배양물로부터 세균을 수득할 수 있다. 수득된 세균은 완충액으로 세척될 수 있다.Methods for obtaining bacteria from culture may be known. Bacteria can be obtained from cultures, for example through centrifugation, filtration, or a combination thereof. The obtained bacteria can be washed with a buffer solution.
상기 방법은 수득된 세균으로부터 지질을 수득하는 단계를 포함한다. 지질을 수득하는 방법은 공지된 것일 수 있다. MLA는 물리적 또는 화학적 방법으로 수득될 수 있다. 물리적 방법은 예를 들어 초음파 또는 냉해동 반복을 수행할 수 있다. 화학적 방법은 유기 용매를 사용하여 추출하는 것일 수 있다. 유기 용매는 예를 들어, 클로로포름, 페놀, 페트롤레움 에테르(petroleum ether), 디클로로메탄(dichloromethane), 메탄올, 헥산, 이소프로필 알콜, 에틸 아세테이트, 아세토니트릴, 에탄올, 부탄올, 또는 이들의 조합일 수 있다. 지질을 추출하는 방법은 예를 들어 블라이와 다이어(Bligh and Dyer) 추출법(Bligh, E.G. and Dyer, W.J., Can. J. Biochem. Physiol., 1959, vol.37, p.911-917)일 수 있다.The method includes obtaining a lipid from the obtained bacteria. The method for obtaining the lipid may be known. MLA can be obtained by physical or chemical methods. The physical method may, for example, perform ultrasonic or cold thaw repetition. The chemical method may be extraction using an organic solvent. The organic solvent may be, for example, chloroform, phenol, petroleum ether, dichloromethane, methanol, hexane, isopropyl alcohol, ethyl acetate, acetonitrile, ethanol, butanol, or a combination thereof. . The method for extracting lipids may be, for example, Bligh and Dyer extraction (Bligh, EG and Dyer, WJ, Can. J. Biochem. Physiol., 1959, vol. 37, p.911-917). have.
상기 방법은 수득된 지질로부터 모노포스포릴 지질 A를 수득하는 단계를 포함한다.The method includes obtaining monophosphoryl lipid A from the obtained lipid.
상기 방법은 수득된 지질로부터 당 모이어티(sugar moiety)를 제거하는 단계를 포함하지 않을 수 있다. 당 모이어티는 Kdo일 수 있다.The method may not include the step of removing a sugar moiety from the obtained lipid. The sugar moiety can be Kdo.
상기 모노포스포릴 지질 A를 수득하는 단계는 크로마토그래피로 수행될 수 있다. 크로마토그래피(chromatography)는 흡착제(고정상 또는 수지(resin))와 시료간의 친화도에 따라 혼합물을 분리하는 것으로서, 용매(이동상 또는 완충액)를 흘려 시료가 용출되도록 하는 방법을 말한다. 상기 크로마토그래피는 이온 교환 크로마토그래피(ion-exchange chromatography), 얇은 막 크로마토그래피(thin layer chromatography: TLC), 액체 크로마토그래피(liquid chromatography: LC), 역상 크로마토그래피(reversed-phase chromatography), 또는 이들의 조합일 수 있다.The step of obtaining the monophosphoryl lipid A may be performed by chromatography. Chromatography refers to a method of separating a mixture according to the affinity between an adsorbent (fixed phase or resin) and a sample, and allowing a sample to be eluted by flowing a solvent (mobile phase or buffer). The chromatography includes ion-exchange chromatography, thin layer chromatography (TLC), liquid chromatography (LC), reverse-phase chromatography, or their It can be a combination.
상기 이온 교환 크로마토그래피는 이온 또는 극성 분자들을 고정상 (또는 수지(resin))에 결합된 음이온 또는 양이온과의 정전기적 인력(electrostatic interaction)을 이용하여 분리하는 방법일 수 있다. 상기 이온 교환 크로마토그래피는 음이온-교환 크로마토그래피 또는 양이온-교환 크로마토그래피일 수 있다. 상기 이온 교환 크로마토그래피의 수지는 예를 들어 셀룰로스, 세파덱스(Sephadex), 또는 세파로스(Sepharose)이다. 상기 음이온-교환 크로마토그래피의 수지는 디에틸아미노에틸(diethylaminoethyl: DEAE)기, 디에틸-2-히드록시프로필아미노에틸(diethyl-2-hydroxypropylaminoethyl 또는 quaternary aminoethyl: QAE)기, 또는 제4급 암모늄(Quaternary ammonium: Q)의 기능기를 갖는 것일 수 있다. 상기 음이온-교환 크로마토그래피의 수지는 DEAE 셀룰로스, QAE 세파덱스, 제4급 암모늄(Quaternary ammonium: Q) 세파로스, 또는 이들의 조합일 수 있다. 상기 음이온-교환 크로마토그래피의 수지는 예를 들어, Macro-prep High Q-3HT, UNO sphere Q, Nuvia Q 레진, 및 DE52 레진이다.The ion exchange chromatography may be a method of separating ions or polar molecules by using electrostatic interaction with anions or cations bound to a stationary phase (or resin). The ion exchange chromatography may be an anion-exchange chromatography or a cation-exchange chromatography. The resin of the ion exchange chromatography is, for example, cellulose, Sephadex, or Sepharose. The resin of the anion-exchange chromatography is a diethylaminoethyl (DEAE) group, a diethyl-2-hydroxypropylaminoethyl (diethyl-2-hydroxypropylaminoethyl or quaternary aminoethyl: QAE) group, or a quaternary ammonium ( It may have a functional group of Quaternary ammonium: Q). The resin of the anion-exchange chromatography may be DEAE cellulose, QAE Sephadex, quaternary ammonium (Q) sepharose, or a combination thereof. The resins of the anion-exchange chromatography are, for example, Macro-prep High Q-3HT, UNO sphere Q, Nuvia Q resin, and DE52 resin.
상기 이온 교환 크로마토그래피에 사용된 완충액(buffer)은 아세트산 암모늄(ammonium acetate), 포름산 암모늄(ammonium formate), 포름산 피리디늄(pyridinium formate), 아세트산 피리디늄(pyridinium acetate), 탄산암모늄(ammonium carbonate), 또는 이들의 조합을 포함할 수 있다. 상기 이온 교환 크로마토그래피에 사용된 용리액(eluent)은 클로로포름, 메탄올, 물, 또는 이들의 조합을 포함할 수 있다.The buffer used in the ion exchange chromatography is ammonium acetate, ammonium formate, pyridinium formate, pyridinium acetate, ammonium carbonate, Or a combination thereof. The eluent used in the ion exchange chromatography may include chloroform, methanol, water, or a combination thereof.
상기 얇은 막 크로마토그래피(thin layer chromatography: TLC)는 유리판이나 알루미늄박 등의 지지체 상에 균일하게 도포된 미립자 운반체를 고정상으로 하고 용매를 이동상으로 하여 물질을 전개 및 분리하는 크로마토그래피를 말한다.The thin layer chromatography (TLC) refers to chromatography in which a material is developed and separated by using a particulate carrier uniformly coated on a support such as a glass plate or aluminum foil as a stationary phase and a solvent as a mobile phase.
상기 액체 크로마토그래피(liquid chromatography: LC)는 이동상으로 액체를 이용하는 크로마토그래피를 말한다. 상기 액체 크로마토그래피는 고성능 액체크로마토그래피(high-performance liquid chromatograph: HPLC)일 수 있다.The liquid chromatography (LC) refers to chromatography using a liquid as a mobile phase. The liquid chromatography may be high-performance liquid chromatography (HPLC).
상기 역상 크로마토그래피(reversed-phase chromatography)는 극성이 작은 고정상과 극성이 큰 이동상의 조합을 사용하여 혼합물을 분리하는 크로마토그래피를 말한다. 상기 역상 크로마토그래피는 실리카겔에 탄화수소 사슬, 페닐기, 시아노기, 아민기, 또는 이들의 조합이 접합된 물질을 고정상으로 이용할 수 있다. 상기 탄화수소 사슬은 C8 사슬, C4 사슬, 또는 C18 사슬일 수 있다.The reversed-phase chromatography refers to chromatography for separating a mixture using a combination of a stationary phase with a small polarity and a mobile phase with a large polarity. In the reverse phase chromatography, a material in which a hydrocarbon chain, a phenyl group, a cyano group, an amine group, or a combination thereof is conjugated to a silica gel may be used as the stationary phase. The hydrocarbon chain may be a C 8 chain, a C 4 chain, or a C 18 chain.
상기 역상 크로마토그래피에 사용된 완충액은 클로로포름, 메탄올, 아세토니트릴, 인산, 암모늄 아세테이트, 물, 또는 이들의 조합을 포함할 수 있다.The buffer used in the reverse phase chromatography may contain chloroform, methanol, acetonitrile, phosphoric acid, ammonium acetate, water, or a combination thereof.
상기 역상 크로마토그래피는 계단식(step) 농도구배 또는 선형(linear) 농도구배를 이용하여 용출되는 것일 수 있다.The reverse phase chromatography may be eluted using a step concentration gradient or a linear concentration gradient.
모노포스포릴 지질 A(MPLA)를 생산하는 세균으로부터 MPLA를 고순도 및 고효율로 생산할 수 있다.MPLA can be produced with high purity and high efficiency from bacteria producing monophosphoryl lipid A (MPLA).
도 1a는 EcLpxL 및 EcLpxM을 포함하는 PCR 산물을 제조하는 방법을 나타내는 모식도이고, 도 1b는 상동 재조합 방법을 이용하여 탈인산화효소를 코딩하는 유전자 lpxE로 대장균 염색체의 bacA를 대체하는 방법을 나타내는 모식도이고, 도 1c는 염색체에 삽입된 탈인산화 효소의 활성을 안정화시킨 균주를 제작하는 과정을 나타낸 모식도이고, 및 도 1d는 KHSC0055 지질의 TLC 결과를 나타내는 사진이다.
도 2는 대장균 KHSC0055의 본배양 세포성장 곡선을 나타낸 그래프이다.
도 3은 대장균 KHSC0055 균주의 배양액으로부터 총 지질을 수득하고 TLC 분석한 결과를 나타내는 사진이다.
도 4a는 대장균 배양 시간에 따른 지질 A와 1-탈아실-지질 A의 TLC 분석 결과를 나타내는 사진이고, 도 4b는 대장균 배양 시간에 따른 지질 중 1-탈인산-지질 A와 지질 A의 비율을 나타낸 그래프이다.
도 5a는 총 지질을 이온교환 크로마토그래피로 정제하고 염 농도구배별 용출액을 TLC로 분석한 결과를 나타내는 사진이고, 도 5b는 DE52 레진 또는 Macro-prep DEAE 레진을 사용하여 정제한 경우 정제된 1-탈인산-지질 A를 TLC로 분석한 결과를 나타내는 사진이다.
도 6은 여러 종류의 이온교환 수지를 사용하여 정제한 경우 정제된 1-탈인산-지질 A를 TLC로 분석한 결과를 나타내는 사진이다.
도 7은 지질 중 인산기 함량의 표준곡선이다.
도 8a는 역상 크로마토그래피에 의한 1-탈인산-지질 A의 정제에서 용출액 별 TLC 분석 결과를 나타내는 사진이고, 도 8b는 정제 단계별 TLC 분석 결과를 나타내는 사진이다.
도 9는 고성능 액체크로마토그래피를 사용하여 DEAE 정제 및 C8 정제 전후의 크로마토그램이다.
도 10a는 선형 농도구배를 통한 C8 정제 후 6 아실 1-탈인산-지질 A의 TLC 분석 결과를 나타내는 사진이고, 도 10b는 HPLC에 의한 1-탈인산-지질 A의 선택적 정제를 나타내는 크로마토그램이다. Figure 1a is a schematic view showing a method for producing a PCR product containing the EcLpxL and EcLpxM, Figure 1b is a schematic diagram illustrating a method for replacing bacA of the E. coli chromosome by gene lpxE encoding a dephosphorylation enzyme by using a homologous recombination method, and , FIG. 1C is a schematic diagram showing a process of preparing a strain stabilizing the activity of a dephosphorylation enzyme inserted into a chromosome, and FIG. 1D is a photograph showing the TLC result of KHSC0055 lipid.
Figure 2 is a graph showing the main culture cell growth curve of E. coli KHSC0055.
3 is a photograph showing the result of TLC analysis obtained by obtaining total lipids from the culture medium of E. coli KHSC0055 strain.
Figure 4a is a photograph showing the result of TLC analysis of lipid A and 1-deacyl-lipid A according to E. coli culture time, and Figure 4b is a ratio of 1-dephosphoric acid-lipid A and lipid A among lipids according to E. coli culture time. This is the graph shown.
Figure 5a is a photograph showing the result of purifying total lipids by ion exchange chromatography and analyzing the eluate according to the salt concentration gradient by TLC, and Figure 5b is purified using DE52 resin or Macro-prep DEAE resin. This is a photograph showing the result of analyzing dephosphorylation-lipid A by TLC.
6 is a photograph showing the results of analyzing purified 1-dephosphoric acid-lipid A by TLC when purified using various types of ion exchange resins.
7 is a standard curve of the content of phosphate groups in lipids.
8A is a photograph showing the result of TLC analysis for each eluate in the purification of 1-dephosphoric acid-lipid A by reverse phase chromatography, and FIG. 8B is a photograph showing the result of TLC analysis at each purification step.
9 is a chromatogram before and after DEAE purification and C8 purification using high performance liquid chromatography.
Figure 10a is a photograph showing the result of TLC analysis of 6 acyl 1-dephosphoric acid-lipid A after C8 purification through a linear concentration gradient, and Figure 10b is a chromatogram showing the selective purification of 1-dephosphoric acid-lipid A by HPLC. .
이하 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.It will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실시예 1. 모노포스포릴 지질 A 생산 균주의 준비Example 1. Preparation of monophosphoryl lipid A producing strain
1.1. 지질화효소 대장균 LpxL 및 대장균 LpxM을 코딩하는 폴리뉴클레오티드를 함유하는 벡터의 준비1.1. Preparation of vectors containing polynucleotides encoding lipolytic enzymes E. coli LpxL and E. coli LpxM
1.1.1. pWSK29-EcLpxLEcLpxM의 준비1.1.1. Preparation of pWSK29-EcLpxLEcLpxM
대장균 LpxL 폴리펩티드를 코딩하는 폴리뉴클레오티드를 수득하기 위해, 대장균 W3110 게놈(GenBank Accession No. NC_000918.1)(ATCC)으로부터 하기 프라이머 쌍을 사용한 제1 중합효소 연쇄 반응(polymerase chain reaction: PCR)을 수행하여, 리보솜 결합 부위(ribosome binding site: RBS)를 포함한 EcLpxL 폴리펩티드(GenBank Accession No. BAA35852.1, 서열번호 1)를 코딩하는 폴리뉴클레오티드(GenBank Accession No. AP009048.1 (c1118159..1117239, 서열번호 2)를 증폭하였다.To obtain a polynucleotide encoding an E. coli LpxL polypeptide, a first polymerase chain reaction (PCR) using the following primer pairs was performed from the E. coli W3110 genome (GenBank Accession No. NC_000918.1) (ATCC). , A polynucleotide encoding an EcLpxL polypeptide (GenBank Accession No. BAA35852.1, SEQ ID NO: 1) including a ribosome binding site (RBS) (GenBank Accession No. AP009048.1 (c1118159..1117239, SEQ ID NO: 2)) ) Was amplified.
LpxL 정방향 프라이머 P1: 5'-CGCAGTCTAGAAAGGAGATATATTGATGACGAATTACCCAAGTLpxL forward primer P1: 5'-CGCAGTCTAGAAAGGAGATATATTGATGACGAATTACCCAAGT
TCTC-3' (서열번호 3)TCTC-3' (SEQ ID NO: 3)
LpxL 역방향 프라이머 P2: 5'-CGCTATTATTTTTTTTCGTTTCCATTGGTATATCTCCTTCTTALpxL reverse primer P2: 5'-CGCTATTATTTTTTTTCGTTTCCATTGGTATATCTCCTTCTTA
TTAATAGCGTGAAGGAACGCCTTC-3' (서열번호 4)TTAATAGCGTGAAGGAACGCCTTC-3' (SEQ ID NO: 4)
대장균 LpxM 폴리펩티드를 코딩하는 폴리뉴클레오티드를 수득하기 위해, 상기 대장균 W3110 게놈으로부터 하기 프라이머 쌍을 사용한 제2 PCR을 수행하여, RBS를 포함한 EcLpxM 폴리펩티드(GenBank Accession No. BAA15663.1, 서열번호 5)를 코딩하는 폴리뉴클레오티드(GenBank Accession No. AP009048.1 (c1941907..1940936, 서열번호 6)를 증폭하였다(도 1a).In order to obtain a polynucleotide encoding an E. coli LpxM polypeptide, a second PCR using the following primer pair was performed from the E. coli W3110 genome to encode an EcLpxM polypeptide (GenBank Accession No. BAA15663.1, SEQ ID NO: 5) including RBS. The polynucleotide (GenBank Accession No. AP009048.1 (c1941907..1940936, SEQ ID NO: 6) was amplified (Fig. 1A).
LpxM 정방향 프라이머 P3: 5'-GAAGGCGTTCCTTCACGCTATTAATAAGAAGGAGATATACCAALpxM forward primer P3: 5'-GAAGGCGTTCCTTCACGCTATTAATAAGAAGGAGATATACCAA
TGGAAACGAAAAAAAATAATAGCG-3' (서열번호 7)TGGAAACGAAAAAAAATAATAGCG-3' (SEQ ID NO: 7)
LpxM 역방향 프라이머 P4: 5'-GCAGAAGCTTTTATTTGATGGGATAAAGATCTTTGCG-3' (서열번호 8)LpxM reverse primer P4: 5'-GCAGAAGCTTTTATTTGATGGGATAAAGATCTTTGCG-3' (SEQ ID NO: 8)
상기 제1 PCR로부터 수득된 EcLpxL 폴리뉴클레오티드 및 제2 PCR로부터 수득된 EcLpxM 폴리뉴클레오티드를 주형으로 하고, 상기 LpxL 정방향 프라이머 P1과 LpxM 역방향 프라이머 P4를 사용한 제3 PCR을 수행하여 EcLpxL 폴리뉴클레오티드와EcLpxM 폴리뉴클레오티드가 융합된 EcLpxLEcLpxM 폴리뉴클레오티드를 증폭하였다.Using the EcLpxL polynucleotide obtained from the first PCR and the EcLpxM polynucleotide obtained from the second PCR as a template, and performing a third PCR using the LpxL forward primer P1 and LpxM reverse primer P4, EcLpxL polynucleotide and EcLpxM polynucleotide The fused EcLpxLEcLpxM polynucleotide was amplified.
상기 PCR을 위해, KOD 핫 스타트 DNA 폴리머라제(Novagen)를 사용하였고, T3000 thermocycler(Biometra)에서 수행하였다.For the PCR, KOD hot start DNA polymerase (Novagen) was used, and it was performed in a T3000 thermocycler (Biometra).
증폭된 산물을 DokDo-Prep PCR purification kit(ELPIS-BIOTECH. Inc.)를 사용하여 정제하고, 정제된 산물을 pWSK29 플라스미드(Wang, R. F., and Kushner, S. R., Gene(1991), vol.100, p.195-199)에 도입하였다. 클로닝된 플라스미드를 대장균 DH5α에 전기천공법(electroporation)으로 형질전환시키고, LB-암피실린 플레이트에서 선별하였다. 클로닝된 플라스미드를 pWSK29-EcLpxLEcLpxM으로 명명하였다(도 1a).The amplified product was purified using a DokDo-Prep PCR purification kit (ELPIS-BIOTECH. Inc.), and the purified product was purified by pWSK29 plasmid (Wang, RF, and Kushner, SR, Gene (1991), vol. 100, p. .195-199). The cloned plasmid was transformed into E. coli DH5α by electroporation, and selected on an LB-ampicillin plate. The cloned plasmid was named pWSK29-EcLpxLEcLpxM (Fig. 1A).
1.1.2. pKHSC0004의 준비1.1.2. Preparation of pKHSC0004
pWSK29-EcLpxLEcLpxM의 프로모터 서열을 PL 프로모터 서열(5'-TTGACATAAATACCACTGGCGGTGATACT-3': 서열번호 9)로 변이시키기 위해, 1.1.1에서 준비된 pWSK29-EcLpxLEcLpxM를 주형으로 하고, 하기 프라이머쌍을 사용하여 부위-특이적 돌연변이유도(site-directed mutagenesis)를 수행하였다.In order to mutate the promoter sequence of pWSK29-EcLpxLEcLpxM to the P L promoter sequence (5'-TTGACATAAATACCACTGGCGGTGATACT-3': SEQ ID NO: 9), pWSK29-EcLpxLEcLpxM prepared in 1.1.1 as a template, and the site- Site-directed mutagenesis was performed.
PL 프로모터 정방향 프라이머: 5'-GGCAGTGAGCGCAACGCAGAATTCTTGACATAAA TACCACTGGCGGTGATACTTTCACACAGGAAACAGCTATGACC-3' (서열번호 10)P L promoter forward primer: 5'-GGCAGTGAGCGCAACGCAGAATTCTTGACATAAA TACCACTGGCGGTGATACTTTCACACAGGAAACAGCTATGACC-3' (SEQ ID NO: 10)
PL 프로모터 역방향 프라이머: 5'-GGTCATAGCTGTTTCCTGTGTGAAAGTATCACCG CCAGTGGTATTTATGTCAAGAATTCTGCGTTGCGCTCACTGCC-3' (서열번호 11)P L promoter reverse primer: 5'-GGTCATAGCTGTTTCCTGTGTGAAAGTATCACCG CCAGTGGTATTTATGTCAAGAATTCTGCGTTGCGCTCACTGCC-3' (SEQ ID NO: 11)
상기 PCR을 위해, Quikchange Site-Directed Mutagenesis Kit(Agilent)을 사용하였고, T3000 thermocycler(Biometra)에서 수행하였다.For the PCR, a Quikchange Site-Directed Mutagenesis Kit (Agilent) was used, and it was performed in a T3000 thermocycler (Biometra).
부위-특이적 돌연변이유도를 수행한 후, 반응물을 Dpn1 제한효소(ELPIS-BIOTECH. Inc.)로 처리하였다. 그 후 대장균 DH5α에 전기천공법(electroporation)으로 형질전환시키고, LB-암피실린 고체 배지에서 선별하였다. 얻어진 플라스미드를 pKHSC0004로 명명하였다.After performing site-specific mutagenesis, the reaction was treated with Dpn1 restriction enzyme (ELPIS-BIOTECH. Inc.). Then, E. coli DH5α was transformed by electroporation and selected in LB-ampicillin solid medium. The resulting plasmid was named pKHSC0004.
1.2. 탈인산화효소를 코딩하는 pBAD30-HpLpxE-frt-kan-frt의 준비1.2. Preparation of pBAD30-HpLpxE-frt-kan-frt encoding dephosphorylation enzyme
1.2.1. pBAD30-HpLpxE의 준비1.2.1. Preparation of pBAD30-HpLpxE
헬리코박터 파이로리 LpxE(Helicobactor pylori LpxE: HpLpxE)를 암호화하는 유전자 hp0021에서, HindIII 제한효소 인식 서열을 없애기 위해, N-말단으로부터 17번째 아미노산인 세린(17Ser)을 코딩하는 뉴클레오티드 서열을 AGC에서 TCG로 돌연변이시켰다. 돌연변이된 hp0021유전자는 Integrated DNA technology(mBiotech, ROK)에서 합성하였다.In the gene hp0021 encoding Helicobacter pylori LpxE (HpLpxE), in order to remove the HindIII restriction enzyme recognition sequence, the nucleotide sequence encoding serine (17Ser), the 17th amino acid from the N-terminus, was mutated from AGC to TCG. . The mutated hp0021 gene was synthesized using Integrated DNA technology (mBiotech, ROK).
돌연변이된 hp0021를 주형으로 하고, 하기 프라이머쌍을 사용하여 HpLpxE 아미노산 서열(서열번호 12)을 코딩하는 폴리뉴클레오티드(서열번호 13)를 증폭하였다.Using the mutated hp0021 as a template, a polynucleotide (SEQ ID NO: 13) encoding the HpLpxE amino acid sequence (SEQ ID NO: 12) was amplified using the following primer pair.
돌연변이 HpLpxE 증폭용 정방향 프라이머: Forward primer for amplification of mutant HpLpxE:
5'-GATCCTCTAGAAAGGAGATATATTGATGAAAAAATTCTTATTTAAACAAAAATTT-3' (서열번호 14)5'-GATCCTCTAGAAAGGAGATATATTGATGAAAAAATTCTTATTTAAACAAAAATTT-3' (SEQ ID NO: 14)
돌연변이 HpLpxE 증폭용 역방향 프라이머: Reverse primer for amplification of mutant HpLpxE:
5'-AGCTACAAGCTTTTAAGGCTTTTTGGGGC-3' (서열번호 15)5'-AGCTACAAGCTTTTAAGGCTTTTTGGGGC-3' (SEQ ID NO: 15)
PCR을 위해, KOD 핫 스타트 DNA 폴리머라제(Novagen)를 사용하였고, T3000 thermocycler(Biometra)에서 수행하였다.For PCR, KOD hot start DNA polymerase (Novagen) was used, and it was performed in a T3000 thermocycler (Biometra).
1.1.1.에 기재된 바와 같이, PCR을 수행하고, 증폭된 산물을 정제하였다. 정제된 산물을 pBAD30(Guzman, L. M., Belin, D., Carson, M. J., Beckwith, J., J Bacteriol (1995). 177(14), p.4121-4130)에 클로닝하였다. 클로닝된 플라스미드를 1.1.1.에 기재된 바와 같이 대장균에 형질전환하고 선별하였다. 클로닝된 플라스미드를 pBAD30-HpLpxE로 명명하였다.As described in 1.1.1., PCR was performed, and the amplified product was purified. The purified product was cloned into pBAD30 (Guzman, L. M., Belin, D., Carson, M. J., Beckwith, J., J Bacteriol (1995). 177(14), p.4121-4130). The cloned plasmid was transformed into E. coli and selected as described in 1.1.1. The cloned plasmid was named pBAD30-HpLpxE.
1.2.2. pBAD30-HpLpxE-frt-kan-frt의 준비1.2.2. Preparation of pBAD30-HpLpxE-frt-kan-frt
HindIII 제한효소 인식 서열을 양쪽에 가진 frt-kan-frt 폴리뉴클레오티드는 하기 프라이머쌍과 pKD4(Kirill A. Datsenko, and Barry L. Wanner PNAS(2000), vol.97, p.6640-6645) 플라스미드를 주형으로 증폭하였다.The frt-kan-frt polynucleotides having HindIII restriction enzyme recognition sequences on both sides of the following primer pairs and pKD4 (Kirill A. Datsenko, and Barry L. Wanner PNAS (2000), vol.97, p.6640-6645) plasmid were used. Amplified as a template.
frt-kan-frt 증폭용 정방향 프라이머:Forward primer for frt-kan-frt amplification:
5'-GCAGAAGCTTGTGTAGGCTGGAGCTGCTTC -3' (서열번호 16)5'-GCAGAAGCTTGTGTAGGCTGGAGCTGCTTC -3' (SEQ ID NO: 16)
frt-kan-frt 증폭용 역방향 프라이머:Reverse primer for frt-kan-frt amplification:
5'-GCAGAAGCTTATGAATATCCTCCTTAGTTCCTAT-3' (서열번호 17)5'-GCAGAAGCTTATGAATATCCTCCTTAGTTCCTAT-3' (SEQ ID NO: 17)
PCR을 위해, pfu DNA 폴리머라제(ELPIS-BIOTECH. Inc.)를 사용하였고, T3000 thermocycler(Biometra)에서 수행하였다. 1.1.1.에 기재된 바와 같이, PCR을 수행하고, 증폭된 산물을 정제하였다.For PCR, pfu DNA polymerase (ELPIS-BIOTECH. Inc.) was used, and performed in a T3000 thermocycler (Biometra). As described in 1.1.1., PCR was performed, and the amplified product was purified.
정제된 산물을 1.2.1에 기재된 바와 같이 수득된 pBAD30-HpLpxE에 클로닝하였다. 클로닝된 플라스미드를 1.1.1에 기재된 바와 같이 대장균에 형질전환하고 선별하였다. 클로닝된 플라스미드를 pBAD30-HpLpxE-frt-kan-frt로 명명하였다.The purified product was cloned into pBAD30-HpLpxE obtained as described in 1.2.1. The cloned plasmid was transformed into E. coli and selected as described in 1.1.1. The cloned plasmid was named pBAD30-HpLpxE-frt-kan-frt.
1.3. 대장균 스트레인의 준비1.3. Preparation of E. coli strain
1.3.1. 1.3.1. lpxTlpxT 유전자가 게놈에서 제거된 대장균의 준비 Preparation of Escherichia coli whose gene has been removed from the genome
대장균 게놈 중 LpxT 폴리펩티드(서열번호 18)를 코딩하는 lpxT 유전자(서열번호 19)에 카나마이신 카세트가 삽입된 대장균 스트레인 lpxT::kan, W3110을 준비하였다. E. coli strains lpxT::kan , W3110 having a kanamycin cassette inserted into the lpxT gene (SEQ ID NO: 19) encoding the LpxT polypeptide (SEQ ID NO: 18) in the E. coli genome were prepared.
pCP20 플라스미드(Kirill A. Datsenko, and Barry L. Wanner PNAS(2000), vol.97, p.6640-6645)를 lpxT::kan, W3110에 형질전환하였다. 선별된 대장균을 LB 고체 배지에 접종하고 42℃의 온도에서 선별하여, lpxT와 카나마이신 카세트가 제거된 대장균 스트레인, 즉 △lpxT, W3110을 준비하였다.The pCP20 plasmid (Kirill A. Datsenko, and Barry L. Wanner PNAS (2000), vol.97, p.6640-6645) was transformed into lpxT::kan, W3110. The selected E. coli was inoculated into LB solid medium and selected at a temperature of 42° C., to prepare an E. coli strain from which lpxT and kanamycin cassette were removed, that is , ΔlpxT, W3110.
1.3.2. 1.3.2. pagPpagP 및 And lpxTlpxT 유전자가 게놈에서 제거된 대장균의 준비 Preparation of Escherichia coli whose gene has been removed from the genome
대장균 게놈 중 pagP 유전자에 카나마이신 카세트가 삽입된 대장균 스트레인 JW0617(pagP::kan)(Keio E.coli knockout library)으로부터 P1 바이러스를 준비하였다. 1.3.1.에서 준비된 △lpxT, W3110에 P1 바이러스를 형질도입시켰다. LB-카나마이신 고체 배지에서 선별하여, pagP 유전자 대신 pagP::kan이 삽입된 대장균 스트레인, 즉 △lpxT, pagP::kan, W3110을 준비하였다. E. coli strain JW0617 (pagP::kan ) (Keio) with a kanamycin cassette inserted into the pagP gene in the E. coli genome E. coli knockout library) was prepared P1 virus. P1 virus was transduced into ΔlpxT , W3110 prepared in 1.3.1. Selected in LB-kanamycin solid medium, pagP::kan instead of the pagP gene was inserted E. coli strains, that is , ΔlpxT, pagP::kan , W3110 was prepared.
1.3.1.에 기재된 바와 같은 pCP20 플라스미드를 상기 △lpxT, pagP::kan, W3110에 형질전환하고 LB-암피실린 고체 배지에서 선별하였다. 선별된 대장균을 LB 고체 배지에 접종하고 42℃의 온도에서 선별하여, pagP와 카나마이신 카세트가 제거된 대장균 스트레인, 즉 △lpxT, △pagP, W3110을 준비하였다.The pCP20 plasmid as described in 1.3.1. was transformed into ΔlpxT, pagP::kan , W3110 and selected in LB-ampicillin solid medium. The selected E. coli was inoculated into LB solid medium and selected at a temperature of 42° C., to prepare E. coli strains from which pagP and kanamycin cassettes were removed, that is , ΔlpxT, ΔpagP, and W3110.
1.3.3. 1.3.3. lpxTlpxT 및 And pagPpagP 유전자가 게놈에서 제거되고 게놈의 The gene is removed from the genome and bacA bacA 유전자가 Gene HpLpxE-frt-kan-frtHpLpxE-frt-kan-frt 폴리뉴클레오티드로 대체된 대장균의 준비 Preparation of Escherichia coli replaced by polynucleotide
bacA 유전자와 상동 재조합을 할 수 있는 bacA::HpLpxE-frt-kan-frt 폴리뉴클레오티드를 준비하기 위해, 1.2.2.에서 준비된 pBAD30-HpLpxE-frt-kan-frt를 주형으로 하고, 하기 프라이머쌍을 이용하여 증폭하였다. To prepare a bacA::HpLpxE-frt-kan-frt polynucleotide capable of homologous recombination with the bacA gene, pBAD30-HpLpxE-frt-kan-frt prepared in 1.2.2 as a template, and the following primer pairs And amplified.
bacA::HpLpxE-frt-kan-frt 증폭용 정방향 프라이머:bacA::HpLpxE-frt-kan-frt amplification Forward Primer:
5'-AACCTGGTCATACGCAGTAGTTCGGACAAGCGGTACATTTTAATAATTTAGGGGTTTATTGATGAAA5'-AACCTGGTCATACGCAGTAGTTCGGACAAGCGGTACATTTTAATAATTTAGGGGTTTATTGATGAAA
AAATTCTTATTTAAACAAAAAT-3' (서열번호 20)AAATTCTTATTTAAACAAAAAT-3' (SEQ ID NO: 20)
bacA::HpLpxE-frt-kan-frt 증폭용 역방향 프라이머:Reverse primer for amplification of bacA::HpLpxE-frt-kan-frt:
5'-TGACAACGCCAAGCATCCGACACTATTCCTCAATTAAAAGAACACGACATACACCGCAGCCGCCACA5'-TGACAACGCCAAGCATCCGACACTATTCCTCAATTAAAAGAACACGACATACACCGCAGCCGCCACA
TGAATATCCTCCTTAGTTCCTA-3' (서열번호 21)TGAATATCCTCCTTAGTTCCTA-3' (SEQ ID NO: 21)
PCR을 위해, pfu DNA 폴리머라제(ELPIS)를 사용하였고, T3000 thermocycler(Biometra)에서 수행하였다.For PCR, pfu DNA polymerase (ELPIS) was used, and performed in a T3000 thermocycler (Biometra).
1.1.1에 기재된 바와 같이, PCR을 수행하고, 증폭된 산물을 정제하였다. 정제된 산물은 대장균 DY330(Yu, D., et. al., PNAS. (2000). 97(11), p5978-5983)에 전기천공법으로 형질전환하였다. 형질전환에 의해, DY330 게놈의 bacA 유전자 위, 아래 염기 서열과의 상동 재조합과정을 통해 bacA::HpLpxE-frt-kan-frt, DY330 대장균을 제조하였다(도 1b).PCR was performed as described in 1.1.1, and the amplified product was purified. The purified product was transformed into E. coli DY330 (Yu, D., et. al., PNAS. (2000). 97(11), p5978-5983) by electroporation. By transformation, bacA ::HpLpxE-frt-kan- frt and DY330 E. coli were prepared through homologous recombination with the nucleotide sequences above and below the bacA gene of the DY330 genome (Fig. 1b).
bacA::HpLpxE-frt-kan-frt, DY330 로부터 P1 바이러스를 준비하였다. 상기 P1 바이러스를 1.3.2.에서 준비된 대장균 △lpxT, △pagP, W3110 스트레인에 형질도입하고 LB-카나마이신 고체 배지에서 선별하였다. 선별된 대장균을 △lpxT, △pagP, bacA::HpLpxE-frt-kan-frt, W3110으로 명명하였다(도 1b).P1 virus was prepared from bacA::HpLpxE-frt-kan-frt, DY330. The P1 virus was transduced into the E. coli △ lpxT , △ pagP , W3110 strain prepared in 1.3.2, and selected in LB-kanamycin solid medium. The selected E. coli was named as △ lpxT, △ pagP , bacA::HpLpxE-frt-kan-frt, W3110 (Fig. 1b).
1.3.4.1.3.4. lpxT lpxT 및 And pagPpagP 유전자가 게놈에서 제거되고 게놈의 The gene is removed from the genome and bacA bacA 유전자가 Gene HpLpxEHpLpxE 유전자로 대체된 대장균의 준비 Preparation of E. coli replaced by gene
1.3.1.에 기재된 바와 같은 pCP20 플라스미드를 1.3.3.에 기재된 바와 같이 준비된 △lpxT, △pagP, bacA::HpLpxE-frt-kan-frt, W3110에 형질전환시키고 LB-암피실린 고체 배지에서 선별하였다. 선별된 대장균을 LB 고체 배지에 접종하고 42℃의 온도에서 선별하여, bacA와 카나마이신 카세트가 제거되고 HpLpxE가 도입된 대장균 스트레인, 즉 △lpxT, △pagP, bacA::HpLpxE, W3110을 준비하였다(도 1c의 첫번째 줄).The pCP20 plasmid as described in 1.3.1. was transformed into Δ lpxT , Δ pagP , bacA::HpLpxE-frt-kan-frt, W3110 prepared as described in 1.3.3, and selected in LB-ampicillin solid medium. . The selected E. coli was inoculated into LB solid medium and selected at a temperature of 42°C, and bacA and kanamycin cassettes were removed, and HpLpxE was introduced into E. coli strains, that is, △ lpxT , △ pagP , bacA::HpLpxE, W3110 were prepared (Fig. 1c, first line).
1.3.5. 1.3.5. lpxTlpxT , , pagP, ybjG pagP, ybjG 유전자가 게놈에서 제거되고 게놈의 The gene is removed from the genome and bacA bacA 유전자가 Gene HpLpxEHpLpxE 유전자로 대체된 대장균의 준비 Preparation of E. coli replaced by gene
대장균 게놈 중 ybjG 유전자에 카나마이신 카세트가 삽입된 대장균 스트레인(JW5112(ybjG::kan))(Keio E.coli knockout library)으로부터 P1 바이러스를 준비하였다. 1.3.4.에서 준비된 △lpxT, △pagP, bacA::HpLpxE, W3110에 P1 바이러스를 형질도입하였다. 대장균을 LB-카나마이신 고체 배지에서 선별하여, ybjG 유전자 대신 ybjG::kan가 삽입된 대장균 스트레인, 즉 △lpxT, △pagP, bacA::HpLpxE, ybjG::kan, W3110을 준비하였다. E. coli strain (JW5112( ybjG::kan )) with a kanamycin cassette inserted into the ybjG gene in the E. coli genome (Keio E. coli knockout library) was prepared P1 virus. P1 virus was transduced into △ lpxT , △ pagP , bacA::HpLpxE, W3110 prepared in 1.3.4. E. coli was selected in LB-kanamycin solid medium, and instead of the ybjG gene, ybjG::kan was inserted into an E. coli strain, that is, △ lpxT , △ pagP , bacA::HpLpxE, ybjG::kan , and W3110.
1.3.1.에 기재된 바와 같은 pCP20 플라스미드를 상기 △lpxT, △pagP, bacA::HpLpxE, ybjG::kan, W3110에 형질전환시키고 LB-암피실린 고체 배지에서 선별하였다. 선별된 대장균을 LB-암피실린 고체 배지에서 선별한 후, 접종하고 42℃의 온도에서 선별하여, ybjG와 카나마이신 카세트가 제거된 대장균 스트레인, 즉 △lpxT, △pagP, △ybjG, bacA::HpLpxE, W3110을 준비하였다(도 1c의 두번째 줄).The pCP20 plasmid as described in 1.3.1. was transformed into the Δ lpxT, Δ pagP , bacA::HpLpxE, ybjG::kan , W3110 and selected in LB-ampicillin solid medium. The selected E. coli was screened in LB-ampicillin solid medium, inoculated and screened at a temperature of 42°C, and the E. coli strain from which ybjG and kanamycin cassette were removed, that is, △ lpxT , △ pagP , △ ybjG, bacA::HpLpxE, W3110 Was prepared (the second line in Fig. 1c).
1.3.6. 대장균 pKHSC0004, △1.3.6. E. coli pKHSC0004, △ lpxTlpxT , △, △ pagPpagP , △, △ ybjG, ybjG, △△ pgpBpgpB , , bacA::HpLpxE, bacA::HpLpxE, W3110 스트레인의 준비Preparation of the W3110 strain
대장균 게놈 중 pgpB 유전자에 카나마이신 카세트가 삽입된 대장균 스트레인(JW1270(pgpB::kan))(Keio E.coli knockout library)으로부터 P1 바이러스를 준비하였다. 1.3.6.에서 준비된 △lpxT, △pagP, △ybjG, bacA::HpLpxE, W3110에 P1 바이러스를 형질도입시키고 LB-카나마이신 고체 배지에서 선별하여, pgpB 유전자 대신 pgpB::kan가 삽입된 대장균 스트레인, 즉 △lpxT, △pagP, △ybjG, bacA::HpLpxE, pgpB::kan W3110을 준비하였다. E. coli strain (JW1270( pgpB::kan )) with a kanamycin cassette inserted into the pgpB gene in the E. coli genome (Keio E. coli knockout library) was prepared P1 virus. 1.3.6. LpxT prepared △, △ pagP, △ ybjG, bacA :: HpLpxE, transduced to P1 virus in W3110 was screened by LB- kanamycin agar, pgpB gene instead of the pgpB :: kan was inserted in the E. coli strain, That is, △ lpxT , △ pagP , △ ybjG, bacA::HpLpxE, pgpB::kan W3110 were prepared.
1.3.1.에 기재된 바와 같은 pCP20 플라스미드를 상기 △lpxT, △pagP, △ybjG, bacA::HpLpxE, pgpB::kan, W3110에 형질전환시키고 LB-암피실린 고체 배지에서 선별하였다. 선별된 대장균을 LB 고체 배지에 접종하고 42℃의 온도에서 선별하여, pgpB와 카나마이신 카세트가 제거된 대장균 스트레인, 즉 △lpxT, △pagP, △ybjG, △pgpB, bacA::HpLpxE, W3110을 준비하였다(도 1c의 세번째 줄).The pCP20 plasmid as described in 1.3.1. was transformed into the △ lpxT, △ pagP , △ ybjG , bacA::HpLpxE, pgpB::kan , W3110 and selected in LB-ampicillin solid medium. The selected E. coli was inoculated into LB solid medium and selected at a temperature of 42°C to prepare an E. coli strain from which pgpB and kanamycin cassettes were removed, that is, △ lpxT , △ pagP , △ ybjG, △ pgpB , bacA::HpLpxE, W3110. (3rd line in Figure 1c).
1.1.2.에서 준비된 pKHSC0004 플라스미드를 준비된 대장균 스트레인 △lpxT, △pagP, △ybjG, △pgpB, bacA::HpLpxE, W3110에 전기천공법으로 형질전환시켰다. 형질전환된 대장균을 LB-암피실린 고체 배지에서 선별하여, 대장균 pKHSC0004, △lpxT, △pagP, △ybjG, △pgpB, bacA::HpLpxE, W3110 스트레인을 준비하였다(도 1c의 네번째 줄).The pKHSC0004 plasmid prepared in 1.1.2 was transformed into the prepared E. coli strain △ lpxT , △ pagP , △ ybjG, △ pgpB , bacA::HpLpxE, W3110 by electroporation. The transformed E. coli was selected in LB-ampicillin solid medium to prepare E. coli pKHSC0004, △ lpxT , △ pagP , △ ybjG , △ pgpB , bacA::HpLpxE, W3110 strain (4th line of FIG. 1c).
1.3.7. 대장균 KHSC0055 스트레인의 준비1.3.7. Preparation of the E. coli KHSC0055 strain
대장균 염색체 중 KdtA 폴리펩티드(서열번호 22)를 코딩하는 kdtA 유전자(서열번호 23)에 카나마이신 카세트(frt-kan-frt)가 삽입되고 pEcKdtA 플라스미드를 포함하고 있는 대장균을 하기 방법으로 준비하였다.In the E. coli chromosome, a kanamycin cassette (frt-kan-frt) was inserted into the kdtA gene (SEQ ID NO: 23) encoding the KdtA polypeptide (SEQ ID NO: 22), and E. coli containing the pEcKdtA plasmid was prepared by the following method.
kdtA 유전자와 상동 재조합할 수 있는 kdtA::frt-kan-frt 폴리뉴클레오티드를 증폭하기 위해, pKD4(Kirill A. Datsenko, and Barry L. Wanner PNAS(2000), vol.97, p.6640-6645) 플라스미드를 주형으로 하고, 하기 프라이머 쌍을 이용하여 증폭하였다. To amplify the kdtA::frt-kan-frt polynucleotide capable of homologous recombination with the kdtA gene, pKD4 (Kirill A. Datsenko, and Barry L. Wanner PNAS (2000), vol.97, p.6640-6645) The plasmid was used as a template, and amplified using the following primer pairs.
kdtA::frt-kan-frt 증폭용 정방향 프라이머:Forward primer for amplification of kdtA::frt-kan-frt:
5'-GCTAAATACATAGAATCCCCAGCACATCCATAAGTCAGCTATTTACTATGCTCGAATTGCGTGTAGG5'-GCTAAATACATAGAATCCCCAGCACATCCATAAGTCAGCTATTTACTATGCTCGAATTGCGTGTAGG
CTGGAGCTGCTTC-3' (서열번호 24)CTGGAGCTGCTTC-3' (SEQ ID NO: 24)
kdtA::frt-kan-frt 증폭용 역방향 프라이머:Reverse primer for amplification of kdtA::frt-kan-frt:
5'-ATCGATATGACCATTGGTAATGGGATCGAAAGTACCCGGATAAATCGCCCGTTTTTGCATTGAATAT5'-ATCGATATGACCATTGGTAATGGGATCGAAAGTACCCGGATAAATCGCCCGTTTTTGCATTGAATAT
CCTCCTTAGTTCCTATTCC-3' (서열번호 25)CCTCCTTAGTTCCTATTCC-3' (SEQ ID NO: 25)
PCR을 위해, pfu DNA 폴리머라제(ELPIS)를 사용하였고, T3000 thermocycler(Biometra)에서 수행하였다.For PCR, pfu DNA polymerase (ELPIS) was used, and performed in a T3000 thermocycler (Biometra).
1.1.1.에 기재된 바와 같이, PCR을 수행하고, 증폭된 산물을 정제하였다. 정제된 산물을 pEcKdtA(Chung, H. S., and Raetz, C. R., Biochemistry(2010), vol.49(19), p.4126-4137)플라스미드를 포함하고 있는 대장균 DY330에 전기천공법으로 형질전환하였다. 형질전환에 의해, DY330 게놈의 kdtA 유전자 위, 아래 염기 서열과의 상동 재조합 과정을 통해 pEcKdtA, kdtA::frt-kan-frt, DY330 대장균을 제조하였다(도 1b). pEcKdtA, kdtA::frt-kan-frt, DY330으로부터 P1 바이러스를 준비하였다. 상기 P1 바이러스를 1.3.6.에서 준비된 대장균 pKHSC0004, △lpxT, △pagP, △ybjG, △pgpB, bacA::HpLpxE, W3110 스트레인에 형질도입하고 LB-카나마이신 고체 배지에서 선별하였다. 선별된 대장균을 KHSC0055(pKHSC0004, △lpxT, △pagP, △ybjG, △pgpB, bacA::HpLpxE, kdtA::frt-kan-frt, W3110)로 명명하였다(도 1c의 5번째 줄).As described in 1.1.1., PCR was performed, and the amplified product was purified. The purified product was transformed into E. coli DY330 containing pEcKdtA (Chung, HS, and Raetz, CR, Biochemistry (2010), vol.49(19), p.4126-4137) plasmid by electroporation. By transformation, pEcKdtA , kdtA::frt-kan- frt, DY330 E. coli were prepared through homologous recombination with nucleotide sequences above and below the kdtA gene of the DY330 genome (Fig. 1b). P1 virus was prepared from pEcKdtA, kdtA::frt-kan-frt, DY330. The P1 virus was transduced into E. coli pKHSC0004, △ lpxT , △ pagP , △ ybjG , △ pgpB , bacA::HpLpxE, W3110 strain prepared in 1.3.6, and selected in LB-kanamycin solid medium. The selected E. coli was named KHSC0055 (pKHSC0004, △ lpxT , △ pagP , △ ybjG , △ pgpB , bacA::HpLpxE, kdtA::frt-kan-frt , W3110) (5th line of FIG. 1c).
1.4. KHSC0055 대장균에서 지질 조성의 확인1.4. KHSC0055 Confirmation of lipid composition in Escherichia coli
1.4.1. KHSC0055의 배양1.4.1. Culture of KHSC0055
1.3.7.에 기재된 바와 같이 대장균 KHSC0055를 준비하였다. KHSC0055 스톡으로부터 50 ㎍/㎖의 암피실린을 함유하는 LB 액체 배지 3 ㎖에 접종하고, 30℃에서 밤새 배양하였다. 배양액을 50 ㎍/㎖의 암피실린을 함유하는 200 ㎖의 LB 액체 배지에 접종하여 30℃에서 밤새 배양하였다.E. coli KHSC0055 was prepared as described in 1.3.7. It was inoculated into 3 ml of LB liquid medium containing 50 µg/ml of ampicillin from KHSC0055 stock, and cultured at 30° C. overnight. The culture solution was inoculated into 200 ml of LB liquid medium containing 50 µg/ml of ampicillin and cultured overnight at 30°C.
1.4.2. KHSC0055 대장균으로부터 지질의 추출1.4.2. KHSC0055 Extraction of lipids from E. coli
1.4.1에 기재된 바와 같이 배양한 대장균 배양액을 상온에서 4000x g의 속도로 약 20분 동안 원심분리하여 각 스트레인의 대장균을 수득하였다. 수득된 대장균을 30 ㎖의 PBS로 세척한 후 8 ㎖의 PBS에 재현탁하였다.The E. coli culture solution cultured as described in 1.4.1 was centrifuged for about 20 minutes at a rate of 4000×g at room temperature to obtain E. coli of each strain. The obtained E. coli was washed with 30 ml of PBS and then resuspended in 8 ml of PBS.
재현탁된 대장균에 10 ㎖의 클로로포름 및 20 ㎖의 메탄올을 가하고, 가끔씩 진탕하면서 약 1 시간 동안 상온에서 인큐베이션하였다. 인큐베이션된 혼합물을 상온에서 2500x g의 속도로 약 30분 동안 원심분리하여 상층액을 수득하였다. 수득된 상층액에 10 ㎖의 클로로포름 및 10 ㎖의 물을 가하고 완전히 혼합한 다음, 상온에서 2500x g의 속도로 약 20분 동안 원심분리하였다. 원심분리된 혼합물에서 유기 용매 층을 분리하고, 상부의 수성 층에 미리 평형화된 유기 용매 층을 첨가하여 유기 용매 층을 2회 추출하였다. 유기 용매 층을 풀링(pooling)하고, 회전 증발기에서 건조시켜 지질을 수득하였다. 수득된 지질을 5 ㎖의 4:1(v/v) 클로로포름:메탄올에 용해시키고, 수조에서 초음파를 조사하였다. 초음파가 조사된 지질을 새로운 시험관으로 옮기고, 수득된 지질을 상온에서 질소 가스 하에서 건조시킨 후 -80℃에서 보관하였다.10 ml of chloroform and 20 ml of methanol were added to the resuspended E. coli, and incubated at room temperature for about 1 hour with occasional shaking. The incubated mixture was centrifuged for about 30 minutes at a rate of 2500x g at room temperature to obtain a supernatant. To the obtained supernatant, 10 ml of chloroform and 10 ml of water were added, mixed thoroughly, and then centrifuged for about 20 minutes at a rate of 2500×g at room temperature. The organic solvent layer was separated from the centrifuged mixture, and the organic solvent layer was extracted twice by adding a pre-equilibrated organic solvent layer to the upper aqueous layer. The organic solvent layer was pooled and dried on a rotary evaporator to give a lipid. The obtained lipid was dissolved in 5 ml of 4:1 (v/v) chloroform:methanol and irradiated with ultrasonic waves in a water bath. The lipids irradiated with ultrasonic waves were transferred to a new test tube, and the obtained lipids were dried under nitrogen gas at room temperature and then stored at -80°C.
1.4.3. 지질의 TLC 분석1.4.3. TLC analysis of lipids
1.4.2.에 기재된 바와 같이 분리된 각 스트레인의 대장균으로부터 막의 지질을 확인하였다. 양성 대조군으로 MPLA Synthetic(InvivoGen, 카탈로그 코드: tlrl-mpls, 합성된 1-탈인산-헥사아실화된 지질 A(도 1d의 레인 1)를 사용하였다.As described in 1.4.2., lipids of the membrane were confirmed from E. coli of each strain separated. As a positive control, MPLA Synthetic (InvivoGen, catalog code: tlrl-mpls, synthesized 1-dephosphoric acid-hexaacylated lipid A (
얇은 막 크로마토그래피(thin layer chromatography: TLC)를 수행하기 위해, 1.4.2.에 기재된 바와 같이 200 ㎖의 대장균 배양액으로부터 지질을 수득하고, 수득된 총 지질의 1/3을 200 ㎕의 4:1(v/v) 클로로포름:메탄올에 용해시켰다. 그 후, 5 ㎕ 내지 15 ㎕를 10 x 10 cm HPTLC 플레이트(EMD Chemicals)에 스폿팅(spotting)하고, 40:25:4:2(v/v)의 클로로포름:메탄올:물:암모늄 히드록시드(28%(v/v) 암모니아)의 용매에서 전개하였다. 전개된 플레이트를 건조시키고, 10%(v/v) 황산(에탄올 중)으로 분무하여 시각화하고, 300℃의 핫 플레이트에서 그을렸다. 지질의 TLC 결과를 도 1d에 나타내었다. 도 1d에서 레인 1은 합성된 1-탈인산-헥사아실화된 지질 A(InvivoGen, 카탈로그 코드 tlrl-mpls)이고, 레인 2는 KHSC0055로부터 분리된 지질이다.To perform thin layer chromatography (TLC), lipids were obtained from 200 ml of E. coli culture medium as described in 1.4.2, and 1/3 of the total lipid obtained was added to 200 μl of 4:1. (v/v) Chloroform: It was dissolved in methanol. Then, 5 μl to 15 μl were spotted on a 10 x 10 cm HPTLC plate (EMD Chemicals), and 40:25:4:2 (v/v) of chloroform:methanol:water:ammonium hydroxide (28% (v/v) ammonia) was developed in a solvent. The developed plate was dried, visualized by spraying with 10% (v/v) sulfuric acid (in ethanol), and smoked on a hot plate at 300°C. The TLC results of lipids are shown in FIG. 1D. In FIG. 1D,
도 1d에 나타난 바와 같이, 대장균 KHSC0055는 1-탈인산-헥사 아실화된 지질 A를 효과적으로 생산하는 살아있는 대장균임을 확인하였다. 따라서, KHSC0055 스트레인은 세포의 생존에 영향을 주지 않으면서 지질다당류(LPS)의 코어-다당류(core-polysaccharide)와 O-항원이 제거되고, 지질 A 및 지질 A에서 인산기 하나가 이미 제거된 모노포스포릴 지질 A가 외막에 직접 축적 및 생산되도록 유전자 조작된 균주이다.As shown in Figure 1d, it was confirmed that E. coli KHSC0055 is a living E. coli that effectively produces 1-dephosphoric acid-hexaacylated lipid A. Therefore, the KHSC0055 strain removes the core-polysaccharide and O-antigen of the lipopolysaccharide (LPS) without affecting the survival of the cells, and the monophosphoric acid group in which one phosphate group has already been removed from lipid A and lipid A. It is a strain genetically engineered to directly accumulate and produce poryl lipid A in the outer membrane.
실시예 2. 모노포스포릴 지질 A의 고순도 또는 고효율 생산 Example 2. High purity or high efficiency production of monophosphoryl lipid A
2.1. 모노포스포릴 지질 A 생산 균주의 배양2.1. Culture of monophosphoryl lipid A producing strain
2.1.1. 배지의 준비2.1.1. Preparation of the medium
실시예 1.4.3.에서 준비된 균주의 배양은 30L 발효조(Fermenter)에서 수행되었으며, 종균 배양 및 본배양 배지를 각각 따로 만들었다. 종배양 배지로는 16.0 g/L의 펩톤(BD Biosciences), 10 g/L의 효모 추출물, 및 5 g/L의 NaCl을 멸균하여 사용하였고, 균주 배양 직전에 100 ㎍/㎖의 암피실린을 첨가하였다. 본배양 배지로는 3.5 g/L의 펩톤(BD Biosciences), 21.0 g/L의 효모 추출물, 6.0 g/L의 KH2PO4, 5.0 g/L의 K2HPO4, 및 5.0 g/L의 NH4Cl을 멸균하여 사용하였고, 40.0 g/L의 글루코스 및 10.0 g/L의 MgSO4·7H2O을 멸균하여 균주를 배양하는 동안 본배양기에 무균적으로 첨가할 수 있게 하였다.The cultivation of the strain prepared in Example 1.4.3. was performed in a 30L fermenter, and seed culture and main culture medium were prepared separately. As a seed culture medium, 16.0 g/L of peptone (BD Biosciences), 10 g/L of yeast extract, and 5 g/L of NaCl were sterilized and used, and 100 μg/ml of ampicillin was added immediately before the strain culture. . As the main culture medium, 3.5 g/L of peptone (BD Biosciences), 21.0 g/L of yeast extract, 6.0 g/L of KH 2 PO 4 , 5.0 g/L of K 2 HPO 4 , and 5.0 g/L of NH 4 Cl was sterilized and used, and 40.0 g/L of glucose and 10.0 g/L of MgSO 4 ·7H 2 O were sterilized to allow the strain to be aseptically added to the main incubator during cultivation.
2.1.2. 종 배양2.1.2. Species culture
1차 종 배양을 위해 1L의 엘렌마이어 플라스크(Erlenmeyer flask)에 200 ㎖의 멸균 종배양 배지를 가하였다. KHSC0055의 종균 바이알을 해동하여 플라스크에 첨가하고 약 31℃의 진탕배양기에서 약 18 내지 약 22시간 동안 배양하였다.For the primary species culture, 200 ml of sterile seed culture medium was added to a 1 L Erlenmeyer flask. The seed vial of KHSC0055 was thawed, added to the flask, and incubated for about 18 to about 22 hours in a shaking incubator at about 31°C.
2차 종 배양을 위해 2L 엘렌마이어 플라스크 6개에 각각 600 ㎖의 멸균 종균배양 배지를 가하고, 35 ㎖의 1차 종균 배양액을 접종하였다. 약 31℃의 진탕배양기에서 약 7 내지 약 12시간 동안 배양하였다.For the secondary species culture, 600 ml of sterilized seed culture medium was added to 6 2L Ellenmeyer flasks, and 35 ml of the primary seed culture solution was inoculated. Incubated for about 7 to about 12 hours in about 31 ℃ shaking incubator.
2.1.3. 본배양2.1.3. Main culture
본배양은 30L 발효기에서 초기 워킹볼륨 18L로 배양하였다. 베셀에 18L의 본배양배지를 채워 멸균하고, 글루코스 용액은 따로 멸균하여 배양하는 동안 무균적으로 배양기에 첨가하였다. 배양액 내의 글루코스의 농도는 1g/L 이하로 조절하였다. 배지의 pH는 NH4OH를 이용하여 pH 6.7을 유지시켜 주었다. 발효기에 2차 종 배양액을 접종하고 약 31℃에서 공기살포(aeration) 3.0 Lpm, 용존 산소량(dissolved oxygen: DO)은 약 50%로 조절하였고 약 300 내지 약 400 rpm으로 교반하면서 배양하였다. 600nm에서 흡광도를 측정하여 배양물의 성장단계를 모니터링하였다(도 2).The main culture was cultured with an initial working volume of 18L in a 30L fermentor. The vessel was sterilized by filling 18 L of the main culture medium, and the glucose solution was separately sterilized and added to the incubator aseptically during cultivation. The concentration of glucose in the culture medium was adjusted to 1 g/L or less. The pH of the medium was maintained at pH 6.7 using NH 4 OH. The second species culture was inoculated into the fermentor, and the aeration at about 31°C was adjusted to 3.0 Lpm and dissolved oxygen (DO) was adjusted to about 50%, and cultured while stirring at about 300 to about 400 rpm. Absorbance was measured at 600 nm to monitor the growth stage of the culture (FIG. 2).
배양액의 회수는 대장균이 지수성장기를 지나 정지기이후 사멸기에 도달하였을 때 회수하였다. 배양액은 원심분리 또는 접선 유동 여과(tangential flow filtration) 중 하나의 방법으로 여과하고 PBS로 세정 후 동결하였다.The culture medium was recovered when the E. coli passed the exponential growth phase and reached the dead phase after the stationary phase. The culture solution was filtered by either centrifugation or tangential flow filtration, washed with PBS, and then frozen.
2.1.4. 지질의 추출 및 박막크로마토그래피(TLC) 분석2.1.4. Lipid extraction and thin-film chromatography (TLC) analysis
배양액을 상온에서 약 20분 동안 원심분리하여 대장균만을 수득하였고, 수득된 대장균 10 ㎖을 40 ㎖의 PBS로 세척한 후 다시 원심분리하여 대장균만을 수득하였다. 세척 2회 반복후 10 ㎖의 PBS에 재현탁 하였다. 재현탁된 대장균 5 ㎖에 메탄올 12.5 ㎖ 및 클로로포름 6.25 ㎖을 가하고 진탕하면서 1시간 동안 상온에서 인큐베이션하였다. 인큐베이션된 혼합물을 상온에서 약 30분간 원심분리하여 상층액을 수득하였다. 수득된 상층액에 메탄올과 클로로포름을 각각 6.25 ㎖씩 취하여 완전히 혼합한 다음 상온에서 20분간 원심분리하였다. 원심분리된 혼합물에서 유기용매 층을 분리하고 질소건조기에서 건조시켜 지질 A와 1-탈인산-지질 A를 추출하였다. 수득된 지질은 냉장보관하였다.The culture solution was centrifuged at room temperature for about 20 minutes to obtain only E. coli, and 10 ml of the obtained E. coli was washed with 40 ml of PBS and then centrifuged again to obtain only E. coli. After repeating the washing twice, it was resuspended in 10 ml of PBS. To 5 ml of the resuspended E. coli, 12.5 ml of methanol and 6.25 ml of chloroform were added, and incubated at room temperature for 1 hour while shaking. The incubated mixture was centrifuged at room temperature for about 30 minutes to obtain a supernatant. 6.25 ml of methanol and chloroform were each added to the obtained supernatant, thoroughly mixed, and then centrifuged at room temperature for 20 minutes. The organic solvent layer was separated from the centrifuged mixture and dried in a nitrogen dryer to extract lipid A and 1-dephosphoric acid-lipid A. The obtained lipid was stored refrigerated.
1.4.3.에 기재된 바와 같이 TLC 분석을 수행하고, 그 결과를 도 3에 나타내었다. 도 3에서 레인 1, 2, 및 3은 1 ㎎/㎖의 수득된 지질을 각각 5, 10, 및 15 ㎕씩 적재(loading)하였다. 도 3에 나타난 바와 같이, 추출된 지질은 지질 A와 1-탈인산-지질 A의 혼합물임을 확인하였다.TLC analysis was performed as described in 1.4.3., and the results are shown in FIG. 3. In FIG. 3,
2.1.5. 세포 성장에 따른 지질의 양 및 조성의 변화2.1.5. Changes in the amount and composition of lipids according to cell growth
대장균의 성장에 따라 총 지질의 양이 어떻게 변화하는지 시험하였다. 2.1.3.의 시간별 배양액 30 ㎖을 샘플링하여 2.1.4에 기지된 바와 같이 지질을 추출하였다. 추출 후 질소건조를 통해 용매를 모두 건조시킨 다음 중량을 측정하였다.It was tested how the amount of total lipids changed according to the growth of E. coli. The lipids were extracted as described in 2.1.4 by sampling 30 ml of the culture solution for each time period of 2.1.3. After extraction, all the solvents were dried through nitrogen drying, and the weight was measured.
표 1에 나타난 바와 같이, 대장균의 세포성장이 정지기(약 21시간 이후)에 도달한 뒤 사멸기까지 총 지질량은 거의 변화가 없었다.As shown in Table 1, after reaching the stoppage (after about 21 hours) of cell growth in Escherichia coli, the total amount of lipids remained almost unchanged until death.
대장균의 성장에 따른 지질 조성의 변화를 확인하기 위해, 추출된 1 ㎎/㎖ 지질을 2:1(v/v)의 클로로포름:메탄올 용액에 용해시켰다. 1.4.3.에 기재된 바와 같이 TLC 분석을 수행하고, 그 결과를 도 4a에 나타내었다. 도 4a에서, 레인 1 내지11은 순서대로 배양 시간 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 및 26시간 배양액으로부터 추출된 지질을 적재하였다. ChemiDoc(BIO-RAD)을 이용하여 1-탈인산-지질 A와 지질 A의 비율을 측정하고, 그 결과를 도 4b에 나타내었다.In order to confirm the change in the lipid composition according to the growth of E. coli, the extracted 1 mg/ml lipid was dissolved in a 2:1 (v/v) chloroform:methanol solution. TLC analysis was performed as described in 1.4.3., and the results are shown in FIG. 4A. In Figure 4a,
도 4a 및 4b에 나타난 바와 같이, 세포성장의 정지기 이후에 지질 A와 1-탈인산-지질 A간에 유의한 비율 변화는 없었다. 살모넬라에서 추출한 1-탈인산-지질 A의 전구체는 배양액의 회수 시간에 따라 LPS의 탈아실 패턴이 변화되어 세포성장 정지기 단계에서 5시간에서 6시간 후 세포를 회수한다고 알려져 있으나(한국 특허번호 10-0884462(2009.02.11)의 청구항 1 참조), KHSC0055의 균주는 세포성장 정지기 이후 회수 시간에 따른 탈아실 패턴의 변화가 거의 없고 배양 시간의 경과에 따라 일정한 비율의 지질 A와 1-탈인산-지질 A가 생성됨을 확인하였다.As shown in Figs. 4a and 4b, there was no significant change in the ratio between lipid A and 1-dephosphoric acid-lipid A after the stationary phase of cell growth. The precursor of 1-dephosphoric acid-lipid A extracted from Salmonella is known to recover cells after 5 to 6 hours in the cell growth stop stage because the deacylation pattern of LPS changes according to the recovery time of the culture medium (Korean Patent No. 10 -0884462 (refer to claim 1 of 2009.02.11)), KHSC0055 strain has little change in the deacylation pattern according to the recovery time after the cell growth stop period, and a constant ratio of lipid A and 1-dephosphoric acid according to the incubation time -It was confirmed that lipid A was generated.
따라서, KHSC0055의 배양액 회수는 세포성장이 정지기에 도달하였을 때 세포를 회수하여 세포의 상태 및 다음 공정인 원심분리 또는 접선 유동 여과의 적용에 더 용이하게 함을 확인하였다.Therefore, it was confirmed that the culture medium recovery of KHSC0055 recovers the cells when the cell growth reaches a stationary phase, and makes it easier to apply the state of the cells and the next step, centrifugation or tangential flow filtration.
2.1.6. 1-탈인산-지질 A의 정제2.1.6. Purification of 1-dephosphoric acid-lipid A
총 지질로부터 1-탈인산-지질 A의 정제를 위하여 이온교환 크로마토그래피를 수행하였다.Ion exchange chromatography was performed for the purification of 1-dephosphoric acid-lipid A from total lipids.
이온교환 크로마토그래피로는 셀룰로스 기질 계열로 IonSep DEAE DE52 Cellulose Preswollen(BiopHoretics Co.)과 폴리머 기질의 Macro Prep DEAE(Bio-Rad Laboratories, Inc.)를 사용하였다. Macro Prep DEAE는 약한 음이온 교환수지로서, -HN+(C2H5)2가 작용기로 작용한다. 해당 정제방법에서 유기용매를 이용하기 때문에 컬럼의 재질은 유리(glass) 또는 스테인레스 스틸을 사용하였다.IonSep DEAE DE52 Cellulose Preswollen (BiopHoretics Co.) as a cellulose substrate and Macro Prep DEAE (Bio-Rad Laboratories, Inc.) as a polymer substrate were used as ion exchange chromatography. Macro Prep DEAE is a weak anion exchange resin, and -HN + (C 2 H 5 ) 2 acts as a functional group. Because the organic solvent is used in the purification method, the material of the column is Glass or stainless steel was used.
지질 A와 1-탈인산-지질 A의 혼합물로부터 1-탈인산-지질 A만을 수득하기위해, 암모늄 아세테이트 농도구배 하에 정제하였다. 클로로포름:메탄올:증류수(2:3:1, v/v)의 용액으로 컬럼 볼륨(Column volume: CV) 10 CV 이상 흘려 레진을 평형화시켰다. 3 mg/㎖의 지질 추출물을 평형화된 레진에 적재하였다. 클로로포름:메탄올:증류수(2:3:1, v/v)의 용액으로 20 CV 이상 레진을 세척하여, 대장균 유래의 불순물들이 충분히 닦여져 용출(elution)되게 하였다.In order to obtain only 1-dephosphoric acid-lipid A from a mixture of lipid A and 1-dephosphoric acid-lipid A, it was purified under an ammonium acetate gradient. The resin was equilibrated by flowing at least 10 CV of a column volume (CV) with a solution of chloroform:methanol:distilled water (2:3:1, v/v). 3 mg/ml of the lipid extract was loaded onto the equilibrated resin. The resin of 20 CV or more was washed with a solution of chloroform:methanol:distilled water (2:3:1, v/v), and impurities derived from E. coli were sufficiently wiped to allow elution.
1-탈인산-지질 A의 용출을 위해, 클로로포름:메탄올: 5 내지 50 mM 염(암모늄 아세테이트)(2:3:1, v/v)의 농도구배를 이용하여 용출하였다. 이때, 1 CV씩 용출된 각 분획에 대해 TLC 분석을 수행하였다(도 5a). TLC 분석결과 1-탈인산-지질 A만 용출된 분획을 수집하여 분별깔대기(separatory funnel)에 풀링하였다. 버퍼 교환 및 1-탈인산-지질 A의 안정성을 높이고자, 풀링된 용액 60 ㎖ 당 10 ㎖의 클로로포름을 가한 후, 0.1 N의 HCl 용액을 가하였다. 이때 최종 클로로포름:메탄올:증류수의 비율은 2:2:1.8(v/v)로 하였다. 분별깔대기를 잘 흔들어 모든 용액이 고르게 섞이도록 한 뒤 2개의 층으로 분리될 때까지 방치하였다. 이때 1-탈인산-지질 A 가 녹아있는 유기용매층과 수용액층이 혼합된 1개 상(phase)에서, 유기용매와 수용액층이 분리된 2개 상으로 상변화가 일어난다. 유기용매층 만을 분리하여 1-탈인산-지질 A를 얻었다. 레진에 여전히 결합되어 있는 지질 A의 용출을 위해 고농도(500 mM 내지 1 M)의 염(암모늄 아세테이트)으로 레진을 재생(Regeneration)시켰다.For elution of 1-dephosphoric acid-lipid A, it was eluted using a concentration gradient of chloroform:methanol: 5 to 50 mM salt (ammonium acetate) (2:3:1, v/v). At this time, TLC analysis was performed on each fraction eluted by 1 CV (FIG. 5A). As a result of TLC analysis, fractions in which only 1-dephosphoric acid-lipid A was eluted were collected and pooled in a separate funnel. To improve the buffer exchange and stability of 1-dephosphoric acid-lipid A, 10 ml of chloroform per 60 ml of the pooled solution was added, followed by 0.1 N HCl solution. At this time, the ratio of chloroform:methanol:distilled water was 2:2:1.8 (v/v). The separatory funnel was shaken well so that all the solutions were evenly mixed, and then left to separate into two layers. At this time, in one phase in which an organic solvent layer in which 1-dephosphoric acid-lipid A is dissolved and an aqueous solution layer are mixed, a phase change occurs into two phases in which the organic solvent and the aqueous solution layer are separated. Only the organic solvent layer was separated to obtain 1-dephosphoric acid-lipid A. The resin was regenerated with a high concentration (500 mM to 1 M) of salt (ammonium acetate) for the elution of lipid A still bound to the resin.
용출액 별 TLC 분석 결과를 도 5a에 나타내었다(레인 1: 정제 전 지질 시료, 레인 2 내지 5: 지질 시료 적재 후 세척, 레인 6 내지 25: 염 농도 구배하여 1 CV 씩 분획(1-탈인산-지질 A 용출), 레인 26 내지 32: 고농도 염으로 세척(지질 A 용출)). 또한, 1-탈인산-지질 A의 정제에서, DE52 레진을 이용한 경우와 Macro-prep DEAE 레진을 이용한 경우를 비교한 결과를 도 5b에 나타내었다.The TLC analysis results for each eluate are shown in FIG. 5A (lane 1: lipid sample before purification,
도 5a 및 도 5b에 나타난 바와 같이, 이온교환 크로마토그래피에 의해 1-탈인산-지질 A를 높은 순도로 정제할 수 있음을 확인하였다.5A and 5B, it was confirmed that 1-dephosphoric acid-lipid A can be purified with high purity by ion exchange chromatography.
2.1.7. 1-탈인산-지질 A 정제를 위한 레진 스크리닝 2.1.7. Resin screening for 1-dephosphoric acid-lipid A purification
DE52, Macro-prep DEAE 레진뿐만 아니라 폴리머 기질 계열의 다른 음이온 교환 수지인 Macro-prep High Q-3HT, UNO sphere Q, Nuvia Q 레진으로도 정제를 시도하였다.Purification was attempted not only with DE52, Macro-prep DEAE resin, but also Macro-prep High Q-3HT, UNO sphere Q, and Nuvia Q resin, which are other anion exchange resins in the polymer matrix series.
1-탈인산-지질 A를 정제하고 TLC로 확인한 결과를 도 6에 나타내었다(레인 1: 정제 전 지질 시료, 레인 2: Macro-prep DEAE를 사용하여 정제한 1-탈인산 지질 A, 레인 3: Macro-prep DEAE 정제 후 재생하여 재사용한 레진으로 정제한 1-탈인산-지질 A, 레인 4: Macro-prep High Q-3HT를 사용 정제한 1-탈인산 지질 A, 레인 5: UNO sphere Q를 사용하여 정제한 1-탈인산 지질 A, 레인 6: Nuvia Q를 사용하여 정제한 1-탈인산 지질 A).1-dephosphoric acid-lipid A was purified and the results confirmed by TLC are shown in FIG. 6 (lane 1: lipid sample before purification, lane 2: 1-dephosphoric acid lipid A purified using Macro-prep DEAE, lane 3) : 1-dephosphoric acid-lipid A purified with regenerated and reused resin after Macro-prep DEAE purification, lane 4: 1-dephosphoric acid lipid A purified using Macro-prep High Q-3HT, lane 5: UNO sphere Q 1-dephosphoric acid lipid A purified using, lane 6: 1-dephosphoric acid lipid A purified using Nuvia Q).
도 6에 나타난 바와 같이, 1-탈인산-지질 A가 지질 추출물로부터 정제되었음을 확인하였다. 또한, Macro-prep DEAE 레진을 이용하여 1-탈인산-지질 A 정제 후 고농도의 염 농도구배로 레진을 재생하였다. 레진 재생이 성공적으로 이루어져, 재생 후에도 1-탈인산-지질 A가 지질 A와의 혼합물로부터 정제되는 것을 확인하였다.As shown in Figure 6, it was confirmed that 1-dephosphoric acid-lipid A was purified from the lipid extract. In addition, after purification of 1-dephosphoric acid-lipid A using Macro-prep DEAE resin, the resin was regenerated with a high salt concentration gradient. Resin regeneration was successful, and it was confirmed that 1-dephosphoric acid-lipid A was purified from a mixture with lipid A even after regeneration.
2.1.8. 지질 A와 1-탈인산-지질 A의 인산기 함량 측정2.1.8. Lipid A and 1-dephosphoric acid-Determination of the phosphate group content of lipid A
지질 A와 1-탈인산-지질 A는 인산기(phosphate)의 개수 차이로 지질 A의 경우 2분자의 인산기를 가지며, 1-탈인산-지질 A는 1분자의 인산기를 가진다. 따라서, 지질 A 1M은 2M의 인산기를 가지며, 1-탈인산-지질 A 1M은 1M의 인산기를 가진다.Lipid A and 1-dephosphoric acid-lipid A have two molecules of phosphate group in the case of lipid A due to the difference in the number of phosphate groups, and 1-dephosphoric acid-lipid A has one molecule of phosphate group. Thus, lipid A 1M has a phosphate group of 2M, and 1-dephosphoric acid-lipid A 1M has a phosphate group of 1M.
KHSC0055의 균주로부터 얻은 지질은 지질 A의 골격에 부착된 지방산들의 갯수 및 위치의 다양성으로 인해 이성질체의 혼합물이다. 수득된 1-탈인산-지질 A의 인산기 함량을 측정하기 위해, 하기 방법을 이용하였다.The lipid obtained from the strain of KHSC0055 is a mixture of isomers due to the diversity of the number and position of fatty acids attached to the backbone of lipid A. In order to measure the phosphate group content of the obtained 1-dephosphoric acid-lipid A, the following method was used.
표준품 0.65 mM Phosphorus standard solution(Sigma cat. no. P3869)을 사용하여 정량곡선을 작성하였다. 0 μmole의 블랭크, 0.0325 μmole(50 ㎕), 0.065 μmole(100 ㎕), 0.114 μmole(175 ㎕), 0.163 μmole(250 ㎕), 및 0.228 μmole(350 ㎕)로 표준품을 각각 유리 시험관에 가하였다. 0.45 ㎖의 8.9 N H2SO4를 각각 시험관에 넣고 200℃ 내지 215℃에서 25분간 인큐베이션하였다. 뜨거워진 유리 시험관을 실온에서 5분간 식힌 후 150 ㎕의 H2O2를 넣고 200℃ 내지 215℃에서 30분간 인큐베이션하였다. 뜨거워진 유리 시험관을 실온에서 식힌 다음 3.9 ㎖의 증류수를 각각의 시험관에 첨가하고 충분히 교반하였다. 각각의 유리 시험관에 0.5 ㎖의 2.5%(w/v) 몰리브덴산 암모늄(Ammonium molybdate)(VI) 4 수화물 용액을 가하고 충분히 교반하였다. 각각의 유리 시험관에 0.5 ㎖의 10%(w/v) 아스코르브산 용액을 가하여 충분히 교반한 다음, 액체가 기화되는 것을 방지하기 위하여 유리 시험관의 뚜껑을 꼭 닫아 100℃에서 7분간 인큐베이션하였다. 시료를 실온에서 식힌 뒤 96웰 플레이트로 옮겼다. ELISA 리더기를 이용하여 820 nm에서 흡광도를 측정하고, 측정한 인산기 함량 표준곡선을 도 7에 나타내었다. 표준 곡선의 R2 값은 0.9989로, 신뢰성이 있다.A quantitative curve was prepared using the standard 0.65 mM Phosphorus standard solution (Sigma cat. no. P3869). Standards were added to a glass test tube with a blank of 0 μmole, 0.0325 μmole (50 μl), 0.065 μmole (100 μl), 0.114 μmole (175 μl), 0.163 μmole (250 μl), and 0.228 μmole (350 μl), respectively. 0.45 ml of 8.9 NH 2 SO 4 was added to each test tube and incubated at 200°C to 215°C for 25 minutes. After cooling the heated glass test tube at room temperature for 5 minutes, 150 µl of H 2 O 2 was added and incubated at 200°C to 215°C for 30 minutes. The heated glass test tube was cooled at room temperature, and then 3.9 ml of distilled water was added to each test tube and sufficiently stirred. 0.5 ml of 2.5% (w/v) ammonium molybdate (VI) tetrahydrate solution was added to each glass test tube and stirred sufficiently. 0.5 ml of 10% (w/v) ascorbic acid solution was added to each glass test tube and sufficiently stirred, and then the lid of the glass test tube was tightly closed to prevent vaporization of the liquid, followed by incubation at 100° C. for 7 minutes. After cooling the sample at room temperature, it was transferred to a 96-well plate. Absorbance was measured at 820 nm using an ELISA reader, and the measured phosphate content standard curve is shown in FIG. 7. The R 2 value of the standard curve is 0.9989, which is reliable.
인산기 함량 표준곡선을 이용하여 정제 전 총 지질, 정제 후 1-탈인산-지질 A 및 지질 A의 인산기 함량을 산출하였다. 산출된 결과를 표 2에 나타내었다.Using the phosphate content standard curve, the total lipid before purification, 1-dephosphoric acid-lipid A after purification, and the phosphate group content of lipid A were calculated. Table 2 shows the calculated results.
(정제후)1-dephosphoric acid-lipid A
(After purification)
(정제후)Lipid A
(After purification)
표 2에 나타난 바와 같이, 정제 전 총 지질의 인산기 함량은 0.91 μmol/㎎이었고, 1-탈인산-지질 A의 인산기 함량은 0.55 μmol/㎎이었고, 지질 A는 그 2배인 1.09 μmol/㎎이었다. 따라서, 정제를 통해 총 지질로부터 1-탈인산-지질 A만 수득되었다는 것을 보여준다.As shown in Table 2, the phosphate content of the total lipid before purification was 0.91 μmol/mg, the phosphate content of 1-dephosphoric acid-lipid A was 0.55 μmol/mg, and lipid A was twice that of 1.09 μmol/mg. Thus, it is shown that only 1-dephosphoric acid-lipid A was obtained from total lipids through purification.
2.1.9. 역상 크로마토그래피에 의한 1-탈인산-지질 A의 정제2.1.9. Purification of 1-dephosphoric acid-lipid A by reverse phase chromatography
이온교환 크로마토그래피를 통해 지질 A로부터 1-탈인산-지질 A의 정제를 수행한 후 남아있는 대장균 유래의 인지질의 분리 정제와 1-탈인산-지질 A의 동종체 함량 조절을 위하여 역상 그로마토그래피(Reverse phase chromatography)를 실시하였다.Reverse phase chromatography for separation and purification of the remaining E. coli-derived phospholipids after purification of 1-dephosphoric acid-lipid A from lipid A through ion exchange chromatography and for controlling the content of 1-dephosphoric acid-lipid A homogenes. (Reverse phase chromatography) was performed.
분리정제를 위한 레진으로 SMT bulk C8 레진(Separation Methods Technologies, Inc.)을 사용하였다. 정제 조건은 용매의 극성도 차이에 의해 진행하였다. 암모늄 아세테이트를 사용하여 시료를 이온화시켰다. 완충액으로 15:75:10(v/v)의 클로로포름:메탄올:증류수(A 버퍼) 및 15:85(v/v)의 클로로포름:메탄올(B 버퍼)를 이용하였다. 두 버퍼에는 모두 20 mM의 암모늄 아세테이트가 녹아져 있다. 정제 방법은 다음과 같다. A 버퍼 100%로 컬럼 볼륨(Column volume: CV) 10 CV 이상 흘려 레진을 평형화시켰다. 이온교환 크로마토그래피를 통해 얻은 1-탈인산-지질 A와 소량의 인지질의 혼합물(시료)을 평형화된 레진에 적재하였다. 이때 혼합물의 농도가 0.5 mg/mL 이하가 되도록 하였다. 그후, A 버퍼 100%로 5 CV만 레진을 세척하여 E.coli 유래의 불순물들이 충분히 닦여져 용출(elution)되게하였다. 이때 4아실 1-탈인산-지질 A도 함께 용출(elution)된다. A 버퍼 95%로 3 CV만 레진을 세척하여 남아있는 불순물을 제거하였다. 이때 소량의 5아실 1-탈인산-지질 A도 용출된다. A 버퍼 50%로 10 CV 이상 흘려서 남아있는 5아실 1-탈인산-지질 A와 6아실 1-탈인산-지질 A를 용출하여 얻을 수 있다. A 버퍼 50% 상태에서 용출된 시료만을 분별깔대기에 풀링하였다. SMT bulk C8 resin (Separation Methods Technologies, Inc.) was used as a resin for separation and purification. Purification conditions were carried out by the difference in the polarity of the solvent. Samples were ionized using ammonium acetate. As a buffer solution, 15:75:10 (v/v) of chloroform:methanol:distilled water (A buffer) and 15:85 (v/v) of chloroform:methanol (B buffer) were used. Both buffers contain 20 mM ammonium acetate. The purification method is as follows. The resin was equilibrated by flowing more than 10 CV of a column volume (CV) with 100% A buffer. A mixture (sample) of 1-dephosphoric acid-lipid A and a small amount of phospholipid obtained through ion exchange chromatography was loaded onto the equilibrated resin. At this time, the concentration of the mixture was made to be 0.5 mg/mL or less. Thereafter, only 5 CV of the resin was washed with 100% A buffer to sufficiently wipe off impurities derived from E. coli to allow elution. At this time, 4-acyl 1-dephosphoric acid-lipid A is also eluted. The remaining impurities were removed by washing only the 3 CV resin with 95% A buffer. At this time, a small amount of 5-acyl 1-dephosphoric acid-lipid A is also eluted. It can be obtained by eluting the remaining 5-acyl 1-dephosphoric acid-lipid A and 6 acyl 1-dephosphoric acid-lipid A by flowing more than 10 CV with 50% A buffer. Only the sample eluted in the state of A
용출액 별 TLC 분석 결과를 도 8a에 나타내었다[레인 1, 10, 및 19: 정제 전 총 지질; 레인 2, 11, 및 30: Macro-prep DEAE를 사용하여 정제한 1-탈인산 지질 A(적재 시료); 레인 3: 통과액(Flow through); 레인 4 내지 8: A 버퍼 100%, 5 CV 분획; 레인 9, 12, 및 13: A 버퍼 5%, 3 CV 분획; 레인 14 내지 18 및 21 내지 25: A버퍼 50%, 10 CV 분획].The results of TLC analysis for each eluate are shown in Fig. 8a [
또한, 정제 단계별 TLC 분석 결과를 도 8b에 나타내었다[레인 1: 정제 전 총 지질; 레인 2: Macro-prep DEAE를 사용하여 정제한 1-탈인산 지질 A(적재 시료); 레인 3: SMT C8 단계 농도구배 정제 후 1-탈인산 지질 A(10 ㎍); 레인 4: SMT C8 단계 농도구배 정제 후 1-탈인산 지질 A(20 ㎍)].In addition, the results of the TLC analysis at each purification step are shown in FIG. 8B [Lane 1: Total lipid before purification; Lane 2: 1-dephosphorylated lipid A (loaded sample) purified using Macro-prep DEAE; Lane 3: 1-dephosphorylated lipid A (10 μg) after SMT C8 step gradient purification; Lane 4: 1-dephosphorylated lipid A (20 µg) after SMT C8 step gradient purification].
도 8a 및 도 8b에 나타난 바와 같이, 6아실acyl 1-탈인산-지질 A가 약 70% 이상인 1-탈인산-지질 A를 수득하였다.As shown in FIGS. 8A and 8B, 1-dephosphoric acid-lipid A having 6 acyl 1-dephosphoric acid-lipid A of about 70% or more was obtained.
버퍼 교환 및 1-탈인산-지질 A의 안정성을 높이고자 암모늄 아세테이트의 중화와 산성 환경의 조성을 위해, 풀링된 용액 60 mL당 10 mL의 클로로포름을 가하였다. 전체 용액의 HCl 농도가 0.1 N이 되도록 HCl과 증류수의 혼합용액을 가하였다. 이때 최종 클로로포름:메탄올:증류수의 비율은 2:2:1.8(v/v)이 되도록 하였다. 깔대기를 잘 흔들어 모든 용액이 고르게 섞이도록 한 뒤 2개의 층으로 분리될 때까지 방치하였다. 1-탈인산-지질 A가 녹아있는 유기용매층과 수용액층이 혼합된 단일 상(single phase)으로부터 유기용매와 수용액층이 분리된 2개의 상(two phase)으로 상변화가 일어났다. 유기용매층 만을 분리하여 1-탈인산-지질 A를 수득하였다.To increase the buffer exchange and stability of 1-dephosphoric acid-lipid A, for neutralization of ammonium acetate and composition of an acidic environment, 10 mL of chloroform was added per 60 mL of the pooled solution. A mixed solution of HCl and distilled water was added so that the HCl concentration of the total solution was 0.1 N. At this time, the ratio of the final chloroform:methanol:distilled water was 2:2:1.8 (v/v). The funnel was shaken well so that all the solutions were evenly mixed, and then left to separate into two layers. A phase change occurred from a single phase in which an organic solvent layer in which 1-dephosphoric acid-lipid A was dissolved and an aqueous solution layer were mixed into two phases in which an organic solvent and an aqueous solution layer were separated. Only the organic solvent layer was separated to obtain 1-dephosphoric acid-lipid A.
2.1.10. 고성능 액체크로마토그래피에 의한 분석2.1.10. Analysis by high performance liquid chromatography
정제 전후의 1-탈인산-지질 A를 역상 고성능 액체크로마토그래피(Reversed phase high-performance liquid chromatograph: RP-HPLC)를 이용하여 분석하였다.1-dephosphoric acid-lipid A before and after purification was analyzed using reversed phase high-performance liquid chromatography (RP-HPLC).
검출기는 charged aerosol detector를 사용하였으며 크로마토그래피 분석은 C8 역상 컬럼 크로마토크래피(Xbridge C8 3.5 ㎛ 4.6 X 250 mm (Waters))를 사용하였다. 시료를 2:1(v/v)의 클로로포름:메탄올에 1 mg/㎖의 농도로 용해시키고, 0.2 ㎛ PTFE 시린지 필터에 통과시켰다. 주입 용적은 5 내지 20 ㎕로 하였다. 암모늄아세테이트와 1%(v/v) 아세트산이 포함된 유기용매에 증류수를 10%(v/v)로 첨가하고, 증류수가 포함되지 않은 버퍼의 농도구배로 분석을 실시하였다. 유속은 0.8 ㎖/분으로 실시하였다.A charged aerosol detector was used as a detector, and a C8 reverse phase column chromatography (Xbridge C8 3.5 µm 4.6 X 250 mm (Waters)) was used for chromatographic analysis. The sample was dissolved in 2:1 (v/v) chloroform:methanol at a concentration of 1 mg/ml, and passed through a 0.2 μm PTFE syringe filter. The injection volume was 5 to 20 µl. Distilled water was added at 10% (v/v) to an organic solvent containing ammonium acetate and 1% (v/v) acetic acid, and the analysis was performed with a concentration gradient of a buffer not containing distilled water. The flow rate was carried out at 0.8 ml/min.
크로마토그래피 정제 전후의 크로마토그램을 비교한 결과를 도 9에 나타내었다. 머무름 시간(Retention time) 약 0분에서 약 6분까지는 기질 피크(matrix peak)이고, 약 6분에서 약 10분까지는 불순물로서 대장균의 세포막을 구성하고 있는 포스파티딜에탄올아민(phosphatidylethanolamine), 포스파티딜-글리세롤(phosphatidyl-glycerol), 카르디오리핀(Cardiolipin) 등의 인지질이 검출되었다. 약 10분에서 약 15분까지 1-탈인산-지질 A, 약 18분에서 약 24분까지 5 아실 사슬의 1-탈인산-지질 A, 약 26분에서 약 30분까지 6 아실 사슬의 1-탈인산-지질 A가 검출되었다.Fig. 9 shows the results of comparing the chromatograms before and after chromatographic purification. Retention time is a matrix peak from about 0 minutes to about 6 minutes, and from about 6 minutes to about 10 minutes as impurities, phosphatidylethanolamine, phosphatidylethanolamine, and phosphatidyl-glycerol ( phosphatidyl-glycerol) and cardiolipin were detected. From about 10 minutes to about 15 minutes 1-Dephosphoric acid-lipid A, from about 18 minutes to about 24
도 9에 나타난 바와 같이, DEAE 정제 결과, 4,5,6 아실의 1-탈인산-지질 A는 약 95%(Relative area)를 차지하였으며, 그중 약 45%가 6 아실 사슬의 1-탈인산-지질 A이었다(맨 위 크로마토그램; DEAE 정제 후 1-탈인산-지질 A).As shown in FIG. 9, as a result of DEAE purification, 1-dephosphoric acid-lipid A of 4,5,6 acyl occupied about 95% (Relative area), of which about 45% was 1-dephosphoric acid of 6 acyl chains. -It was lipid A (top chromatogram; 1-dephosphoric acid-lipid A after DEAE purification).
해당 DEAE Elution 중간체를 이용하여 C8 정제를 실시하였고 크로매토그램 결과를 도 9에 나타내었다(아래 4개의 크로마토그램: C8 정제 후 1-탈인산-지질 A). C8 purification was performed using the DEAE Elution intermediate, and the chromatogram results are shown in FIG. 9 (four chromatograms below: 1-dephosphoric acid-lipid A after C8 purification).
도 9에 나타난 바와 같이, C8 정제를 통해 인지질의 불순물과 4 아실 1-탈인산-지질 A가 대부분 제거되고, 5 아실 1-탈인산-지질 A와 6 아실 1-탈인산-지질 A의 함량이 약 97% 내지 약 98%인 1-탈인산-지질 A를 얻을 수 있었다. As shown in FIG. 9, impurities of phospholipids and 4 acyl 1-dephosphoric acid-lipid A are mostly removed through C8 purification, and the content of 5 acyl 1-dephosphoric acid-lipid A and 6 acyl 1-dephosphoric acid-lipid A From about 97% to about 98%, 1-dephosphoric acid-lipid A could be obtained.
2.1.11. 1-탈인산-지질 A의 선택적 정제2.1.11. 1-dephosphoric acid-selective purification of lipid A
역상 크로마토그래피에서 A 버퍼와 B 버퍼의 선형 농도구배(linear gradient)를 통해 6 아실 1-탈인산-지질 A를 선택적으로 얻을 수 있었다. A 버퍼는 클로로포롬:메탄올:증류수의 비율이 15:75:10(v/v)의 비율로 존재한다. B 버퍼는 A 버퍼보다 더 극성으로 클로로포름: 메탄올의 비율이 15:85(v/v)로 구성되어있으며 모두 5 mM 내지 20 mM 암모늄 아세테이트를 포함한다.In reverse phase chromatography, 6 acyl 1-dephosphoric acid-lipid A could be selectively obtained through a linear gradient of buffer A and buffer B. Buffer A is present in a ratio of chloroform:methanol:distilled water at a ratio of 15:75:10 (v/v). Buffer B is more polar than buffer A, and has a chloroform:methanol ratio of 15:85 (v/v), and all contain 5 mM to 20 mM ammonium acetate.
A 버퍼 100%로 시료를 레진에 적재하고, 5 CV(Column volume)로 레진을 세척하였다. 이때, 이온교환크로마토그래피 후 잔존할 수 있는 대장균 유래의 인지질과 4 아실 1-탈인산-지질 A이 용출되었다. 그 후, B 버퍼 5%에서 B 버퍼 95%까지 선형 농도구배(linear gradient)를 통해 1-탈인산-지질 A를 용출시켰다.A sample was loaded on the resin with 100% buffer A, and the resin was washed with 5 CV (Column volume). At this time, the E. coli-derived phospholipid and 4 acyl 1-dephosphoric acid-lipid A that can remain after ion exchange chromatography were eluted. Then, 1-dephosphoric acid-lipid A was eluted through a linear gradient from 5% B buffer to 95% B buffer.
선형 농도구배를 통한 C8 정제 후 6 아실 1-탈인산-지질 A의 TLC 분석 결과를 도 10a에 나타내었다[레인 1: 정제 전 총 지질; 레인 2: Macro-prep DEAE를 사용하여 정제한 1-탈인산 지질 A(적재 시료); 레인 3: SMT C8 선형 농도구배 정제 후 1-탈인산 지질 A(5 ㎍); 레인 4: SMT C8 선형 농도구배 정제 후 1-탈인산 지질 A(10 ㎍); 레인 5: SMT C8 선형 농도구배 정제 후 1-탈인산 지질 A(20 ㎍)].The results of TLC analysis of 6 acyl 1-dephosphoric acid-lipid A after C8 purification through a linear concentration gradient are shown in FIG. 10A [lane 1: total lipid before purification; Lane 2: 1-dephosphorylated lipid A (loaded sample) purified using Macro-prep DEAE; Lane 3: 1-dephosphorylated lipid A (5 μg) after SMT C8 linear gradient purification; Lane 4: 1-dephosphorylated lipid A (10 μg) after SMT C8 linear gradient purification; Lane 5: 1-dephosphorylated lipid A (20 μg) after SMT C8 linear gradient purification].
또한, HPLC에 의한 1-탈인산-지질 A의 선택적 정제를 나타내는 크로마토그램을 도 10b에 나타내었다[위 크로마토그램: DEAE 정제 후 1-탈인산-지질 A, 아래 크로마토그램: C8로 6acyl의 1-탈인산-지질 A의 선택적 정제].In addition, a chromatogram showing the selective purification of 1-dephosphoric acid-lipid A by HPLC is shown in FIG. 10B [Chromatogram above: 1-dephosphoric acid-lipid A after DEAE purification, lower chromatogram: 1 of 6acyl as C8 -Dephosphoric acid-selective purification of lipid A].
따라서, 1-탈인산-지질 A를 세포막에 축적하는 유전자-변형된 대장균 균주를 배양하고 균주로부터 1-탈인산 지질 A를 고순도로 정제하는 공정을 확보하였다. Accordingly, a gene-modified E. coli strain that accumulates 1-dephosphoric acid-lipid A in the cell membrane was cultured, and a process of purifying 1-dephosphoric acid lipid A with high purity from the strain was obtained.
<110> EUBIOLOGICS CO., LTD. Korea Institute of Science and Technology <120> Method for producing phosphoryl lipid A <130> PN132814KR <150> KR 10-2019-0106640 <151> 2019-08-29 <160> 25 <170> KopatentIn 2.0 <210> 1 <211> 306 <212> PRT <213> Artificial Sequence <220> <223> Escherichia coli LpxL polypeptide <400> 1 Met Thr Asn Leu Pro Lys Phe Ser Thr Ala Leu Leu His Pro Arg Tyr 1 5 10 15 Trp Leu Thr Trp Leu Gly Ile Gly Val Leu Trp Leu Val Val Gln Leu 20 25 30 Pro Tyr Pro Val Ile Tyr Arg Leu Gly Cys Gly Leu Gly Lys Leu Ala 35 40 45 Leu Arg Phe Met Lys Arg Arg Ala Lys Ile Val His Arg Asn Leu Glu 50 55 60 Leu Cys Phe Pro Glu Met Ser Glu Gln Glu Arg Arg Lys Met Val Val 65 70 75 80 Lys Asn Phe Glu Ser Val Gly Met Gly Leu Met Glu Thr Gly Met Ala 85 90 95 Trp Phe Trp Pro Asp Arg Arg Ile Ala Arg Trp Thr Glu Val Ile Gly 100 105 110 Met Glu His Ile Arg Asp Val Gln Ala Gln Lys Arg Gly Ile Leu Leu 115 120 125 Val Gly Ile His Phe Leu Thr Leu Glu Leu Gly Ala Arg Gln Phe Gly 130 135 140 Met Gln Glu Pro Gly Ile Gly Val Tyr Arg Pro Asn Asp Asn Pro Leu 145 150 155 160 Ile Asp Trp Leu Gln Thr Trp Gly Arg Leu Arg Ser Asn Lys Ser Met 165 170 175 Leu Asp Arg Lys Asp Leu Lys Gly Met Ile Lys Ala Leu Lys Lys Gly 180 185 190 Glu Val Val Trp Tyr Ala Pro Asp His Asp Tyr Gly Pro Arg Ser Ser 195 200 205 Val Phe Val Pro Leu Phe Ala Val Glu Gln Ala Ala Thr Thr Thr Gly 210 215 220 Thr Trp Met Leu Ala Arg Met Ser Gly Ala Cys Leu Val Pro Phe Val 225 230 235 240 Pro Arg Arg Lys Pro Asp Gly Lys Gly Tyr Gln Leu Ile Met Leu Pro 245 250 255 Pro Glu Cys Ser Pro Pro Leu Asp Asp Ala Glu Thr Thr Ala Ala Trp 260 265 270 Met Asn Lys Val Val Glu Lys Cys Ile Met Met Ala Pro Glu Gln Tyr 275 280 285 Met Trp Leu His Arg Arg Phe Lys Thr Arg Pro Glu Gly Val Pro Ser 290 295 300 Arg Tyr 305 <210> 2 <211> 921 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Escherichia coli LpxL polypeptide <400> 2 atgacgaatc tacccaagtt ctccaccgca ctgcttcatc cgcgttattg gttaacctgg 60 ttgggtattg gcgtactttg gttagtcgtg caattgccct acccggttat ctaccgcctc 120 ggttgtggat taggaaaact ggcgttacgt tttatgaaac gacgcgcaaa aattgtgcat 180 cgcaacctgg aactgtgctt cccggaaatg agcgaacaag aacgccgtaa aatggtggtg 240 aagaatttcg aatccgttgg catgggcctg atggaaaccg gcatggcgtg gttctggccg 300 gaccgccgaa tcgcccgctg gacggaagtg atcggcatgg aacacattcg tgacgtgcag 360 gcgcaaaaac gcggcatcct gttagttggc atccattttc tgacactgga gctgggtgcg 420 cggcagtttg gtatgcagga accgggtatt ggcgtttatc gcccgaacga taatccactg 480 attgactggc tacaaacctg gggccgtttg cgctcaaata aatcgatgct cgaccgcaaa 540 gatttaaaag gcatgattaa agccctgaaa aaaggcgaag tggtctggta cgcaccggat 600 catgattacg gcccgcgctc aagcgttttc gtcccgttgt ttgccgttga gcaggctgcg 660 accacgaccg gaacctggat gctggcacgg atgtccggcg catgtctggt gcccttcgtt 720 ccacgccgta agccagatgg caaagggtat caattgatta tgctgccgcc agagtgttct 780 ccgccactgg atgatgccga aactaccgcc gcgtggatga acaaagtggt cgaaaaatgc 840 atcatgatgg caccagagca gtatatgtgg ttacaccgtc gctttaaaac acgcccggaa 900 ggcgttcctt cacgctatta a 921 <210> 3 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> LpxL forward primer P1 <400> 3 cgcagtctag aaaggagata tattgatgac gaatctaccc aagttctc 48 <210> 4 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> LpxL reverse primer P2 <400> 4 cgctattatt ttttttcgtt tccattggta tatctccttc ttattaatag cgtgaaggaa 60 cgccttc 67 <210> 5 <211> 323 <212> PRT <213> Artificial Sequence <220> <223> Escherichia coli LpxM polypeptide <400> 5 Met Glu Thr Lys Lys Asn Asn Ser Glu Tyr Ile Pro Glu Phe Asp Lys 1 5 10 15 Ser Phe Arg His Pro Arg Tyr Trp Gly Ala Trp Leu Gly Val Ala Ala 20 25 30 Met Ala Gly Ile Ala Leu Thr Pro Pro Lys Phe Arg Asp Pro Ile Leu 35 40 45 Ala Arg Leu Gly Arg Phe Ala Gly Arg Leu Gly Lys Ser Ser Arg Arg 50 55 60 Arg Ala Leu Ile Asn Leu Ser Leu Cys Phe Pro Glu Arg Ser Glu Ala 65 70 75 80 Glu Arg Glu Ala Ile Val Asp Glu Met Phe Ala Thr Ala Pro Gln Ala 85 90 95 Met Ala Met Met Ala Glu Leu Ala Ile Arg Gly Pro Glu Lys Ile Gln 100 105 110 Pro Arg Val Asp Trp Gln Gly Leu Glu Ile Ile Glu Glu Met Arg Arg 115 120 125 Asn Asn Glu Lys Val Ile Phe Leu Val Pro His Gly Trp Ala Val Asp 130 135 140 Ile Pro Ala Met Leu Met Ala Ser Gln Gly Gln Lys Met Ala Ala Met 145 150 155 160 Phe His Asn Gln Gly Asn Pro Val Phe Asp Tyr Val Trp Asn Thr Val 165 170 175 Arg Arg Arg Phe Gly Gly Arg Leu His Ala Arg Asn Asp Gly Ile Lys 180 185 190 Pro Phe Ile Gln Ser Val Arg Gln Gly Tyr Trp Gly Tyr Tyr Leu Pro 195 200 205 Asp Gln Asp His Gly Pro Glu His Ser Glu Phe Val Asp Phe Phe Ala 210 215 220 Thr Tyr Lys Ala Thr Leu Pro Ala Ile Gly Arg Leu Met Lys Val Cys 225 230 235 240 Arg Ala Arg Val Val Pro Leu Phe Pro Ile Tyr Asp Gly Lys Thr His 245 250 255 Arg Leu Thr Ile Gln Val Arg Pro Pro Met Asp Asp Leu Leu Glu Ala 260 265 270 Asp Asp His Thr Ile Ala Arg Arg Met Asn Glu Glu Val Glu Ile Phe 275 280 285 Val Gly Pro Arg Pro Glu Gln Tyr Thr Trp Ile Leu Lys Leu Leu Lys 290 295 300 Thr Arg Lys Pro Gly Glu Ile Gln Pro Tyr Lys Arg Lys Asp Leu Tyr 305 310 315 320 Pro Ile Lys <210> 6 <211> 972 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Escherichia coli LpxM polypeptide <400> 6 atggaaacga aaaaaaataa tagcgaatac attcctgagt ttgataaatc ctttcgccac 60 ccgcgctact ggggagcatg gctgggcgta gcagcgatgg cgggtatcgc tttaacgccg 120 ccaaagttcc gtgatcccat tctggcacgg ctgggacgtt ttgccggacg actgggaaaa 180 agctcacgcc gtcgtgcgtt aatcaatctg tcgctctgct ttccagaacg tagtgaagct 240 gaacgcgaag cgattgttga tgagatgttt gccaccgcgc cgcaagcgat ggcaatgatg 300 gctgagttgg caatacgcgg gccggagaaa attcagccgc gcgttgactg gcaagggctg 360 gagatcatcg aagagatgcg gcgtaataac gagaaagtta tctttctggt gccgcacggt 420 tgggccgtcg atattcctgc catgctgatg gcctcgcaag ggcagaaaat ggcagcgatg 480 ttccataatc agggcaaccc ggtttttgat tatgtctgga acacggtgcg tcgtcgcttt 540 ggcggtcgtc tgcatgcgag aaatgacggt attaaaccat tcatccagtc ggtacgtcag 600 gggtactggg gatattattt acccgatcag gatcatggcc cagagcacag cgaatttgtg 660 gatttctttg ccacctataa agcgacgttg cccgcgattg gtcgtttgat gaaagtgtgc 720 cgtgcgcgcg ttgtaccgct gtttccgatt tatgatggca agacgcatcg tctgacgatt 780 caggtgcgcc caccgatgga tgatctgtta gaggcggatg atcatacgat tgcgcggcgg 840 atgaatgaag aagtcgagat ttttgttggt ccgcgaccag aacaatacac ctggatacta 900 aaattgctga aaactcgcaa accgggcgaa atccagccgt ataagcgcaa agatctttat 960 cccatcaaat aa 972 <210> 7 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> LpxM forward primer P3 <400> 7 gaaggcgttc cttcacgcta ttaataagaa ggagatatac caatggaaac gaaaaaaaat 60 aatagcg 67 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> LpxM reverse primer P3 <400> 8 gcagaagctt ttatttgatg ggataaagat ctttgcg 37 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PL promoter nucleotide sequence <400> 9 ttgacataaa taccactggc ggtgatact 29 <210> 10 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Forward primer amplifying PL promoter <400> 10 ggcagtgagc gcaacgcaga attcttgaca taaataccac tggcggtgat actttcacac 60 aggaaacagc tatgacc 77 <210> 11 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer amplifying PL promoter <400> 11 ggtcatagct gtttcctgtg tgaaagtatc accgccagtg gtatttatgt caagaattct 60 gcgttgcgct cactgcc 77 <210> 12 <211> 190 <212> PRT <213> Artificial Sequence <220> <223> Helicobactor pylori LpxE mutant <400> 12 Met Lys Lys Phe Leu Phe Lys Gln Lys Phe Cys Glu Ser Leu Pro Lys 1 5 10 15 Ser Phe Ser Lys Thr Leu Leu Ala Leu Ser Leu Gly Leu Ile Leu Leu 20 25 30 Gly Ile Phe Ala Pro Phe Pro Lys Val Pro Lys Gln Pro Ser Val Pro 35 40 45 Leu Met Phe His Phe Thr Glu His Tyr Ala Arg Phe Ile Pro Thr Ile 50 55 60 Leu Ser Val Ala Ile Pro Leu Ile Gln Arg Asp Ala Val Gly Leu Phe 65 70 75 80 Gln Val Ala Asn Ala Ser Ile Ala Thr Thr Leu Leu Thr His Thr Thr 85 90 95 Lys Arg Ala Leu Asn His Val Thr Ile Asn Asp Gln Arg Leu Gly Glu 100 105 110 Arg Pro Tyr Gly Gly Asn Phe Asn Met Pro Ser Gly His Ser Ser Met 115 120 125 Val Gly Leu Ala Val Ala Phe Leu Met Arg Arg Tyr Ser Phe Lys Lys 130 135 140 Tyr Phe Trp Leu Leu Pro Leu Val Pro Leu Thr Met Leu Ala Arg Ile 145 150 155 160 Tyr Leu Asp Met His Thr Ile Gly Ala Val Leu Thr Gly Leu Gly Val 165 170 175 Gly Met Leu Cys Val Ser Leu Phe Thr Ser Pro Lys Lys Pro 180 185 190 <210> 13 <211> 573 <212> DNA <213> Artificial Sequence <220> <223> Polynucleodide encoding Helicobactor pylori LpxE mutant <400> 13 atgaaaaaat tcttatttaa acaaaaattt tgtgaaagcc tgcccaaatc gttttctaaa 60 actttgttag cgctcagttt gggcttgatt ttattaggca tttttgcgcc tttccctaaa 120 gtccctaaac agcctagcgt gcctttaatg tttcatttca ccgagcatta tgcgcgcttt 180 atccctacga ttttatctgt ggcgattccc ttaatccaaa gagatgcggt agggcttttt 240 caagtcgcta acgcttctat cgctacaacc cttctcacgc acaccaccaa aagagcctta 300 aaccatgtaa caatcaacga tcagcgtttg ggcgagcgcc cttatggagg taatttcaac 360 atgccaagcg ggcattcgtc tatggtgggt ttggcggtgg cgtttttaat gcgccgctat 420 tcttttaaaa aatacttttg gctcttgccc ctagtccctt tgaccatgct cgctcgcatt 480 tatttagaca tgcacaccat tggcgcggtg ctgaccgggc ttggcgttgg aatgttgtgc 540 gtaagccttt ttacaagccc caaaaagcct taa 573 <210> 14 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying HpLpxE mutant <400> 14 gatcctctag aaaggagata tattgatgaa aaaattctta tttaaacaaa aattt 55 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying Helicobactor pylori LpxE mutant <400> 15 agctacaagc ttttaaggct ttttggggc 29 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying frt-kan-frt <400> 16 gcagaagctt gtgtaggctg gagctgcttc 30 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying frt-kan-frt <400> 17 gcagaagctt atgaatatcc tccttagttc ctat 34 <210> 18 <211> 237 <212> PRT <213> Artificial Sequence <220> <223> Escherichia coli LpxT polypeptide <400> 18 Met Ile Lys Asn Leu Pro Gln Ile Val Leu Leu Asn Ile Val Gly Leu 1 5 10 15 Ala Leu Phe Leu Ser Trp Tyr Ile Pro Val Asn His Gly Phe Trp Leu 20 25 30 Pro Ile Asp Ala Asp Ile Phe Tyr Phe Phe Asn Gln Lys Leu Val Glu 35 40 45 Ser Lys Ala Phe Leu Trp Leu Val Ala Leu Thr Asn Asn Arg Ala Phe 50 55 60 Asp Gly Cys Ser Leu Leu Ala Met Gly Met Leu Met Leu Ser Phe Trp 65 70 75 80 Leu Lys Glu Asn Ala Pro Gly Arg Arg Arg Ile Val Ile Ile Gly Leu 85 90 95 Val Met Leu Leu Thr Ala Val Val Leu Asn Gln Leu Gly Gln Ala Leu 100 105 110 Ile Pro Val Lys Arg Ala Ser Pro Thr Leu Thr Phe Thr Asp Ile Asn 115 120 125 Arg Val Ser Glu Leu Leu Ser Val Pro Thr Lys Asp Ala Ser Arg Asp 130 135 140 Ser Phe Pro Gly Asp His Gly Met Met Leu Leu Ile Phe Ser Ala Phe 145 150 155 160 Met Trp Arg Tyr Phe Gly Lys Val Ala Gly Leu Ile Ala Leu Ile Ile 165 170 175 Phe Val Val Phe Ala Phe Pro Arg Val Met Ile Gly Ala His Trp Phe 180 185 190 Thr Asp Ile Ile Val Gly Ser Met Thr Val Ile Leu Ile Gly Leu Pro 195 200 205 Trp Val Leu Leu Thr Pro Leu Ser Asp Arg Leu Ile Thr Phe Phe Asp 210 215 220 Lys Ser Leu Pro Gly Lys Asn Lys His Phe Gln Asn Lys 225 230 235 <210> 19 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Escherichia coli LpxT polypeptide <400> 19 atgattaaaa atttgccgca aatagtgttg ttgaatattg tcggcctcgc gctgtttctt 60 tcctggtata tccccgttaa tcatggattc tggttgccga ttgatgcgga tattttttat 120 ttctttaatc agaaactggt cgaaagtaag gcctttttgt ggctggttgc attgaccaac 180 aatcgcgcct tcgacggttg ttcactgctg gcgatgggta tgttgatgct gagtttctgg 240 ctgaaagaaa acgcccctgg cagacgacgt atcgtgatta ttggtctggt catgctatta 300 actgcagtgg tattaaacca gctgggtcag gcattaattc ctgtaaaacg ggccagccca 360 acattgactt ttaccgatat taaccgcgtc agcgaactgc tctctgttcc cacgaaagat 420 gcctcacgag atagctttcc cggcgatcac ggcatgatgc tgcttatttt ttcggcattc 480 atgtggcgtt atttcggcaa agttgcaggc cttatcgccc ttattatttt tgtggttttt 540 gcatttccca gagtaatgat tggcgcacac tggtttactg acatcattgt cggttcgatg 600 accgtgatat tgatcggttt gccctgggtg ttgctgacgc cattaagtga tcgattaatc 660 accttttttg acaaatcact accaggaaaa aacaaacatt tccaaaacaa ataa 714 <210> 20 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Forward primer amplifying bacA::HpLpxE-frt-kan-frt <400> 20 aacctggtca tacgcagtag ttcggacaag cggtacattt taataattta ggggtttatt 60 gatgaaaaaa ttcttattta aacaaaaat 89 <210> 21 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer amplifying bacA::HpLpxE-frt-kan-frt <400> 21 tgacaacgcc aagcatccga cactattcct caattaaaag aacacgacat acaccgcagc 60 cgccacatga atatcctcct tagttccta 89 <210> 22 <211> 425 <212> PRT <213> Artificial Sequence <220> <223> Escherichia coli KdtA polypeptide <400> 22 Met Leu Glu Leu Leu Tyr Thr Ala Leu Leu Tyr Leu Ile Gln Pro Leu 1 5 10 15 Ile Trp Ile Arg Leu Trp Val Arg Gly Arg Lys Ala Pro Ala Tyr Arg 20 25 30 Lys Arg Trp Gly Glu Arg Tyr Gly Phe Tyr Arg His Pro Leu Lys Pro 35 40 45 Gly Gly Ile Met Leu His Ser Val Ser Val Gly Glu Thr Leu Ala Ala 50 55 60 Ile Pro Leu Val Arg Ala Leu Arg His Arg Tyr Pro Asp Leu Pro Ile 65 70 75 80 Thr Val Thr Thr Met Thr Pro Thr Gly Ser Glu Arg Val Gln Ser Ala 85 90 95 Phe Gly Lys Asp Val Gln His Val Tyr Leu Pro Tyr Asp Leu Pro Asp 100 105 110 Ala Leu Asn Arg Phe Leu Asn Lys Val Asp Pro Lys Leu Val Leu Ile 115 120 125 Met Glu Thr Glu Leu Trp Pro Asn Leu Ile Ala Ala Leu His Lys Arg 130 135 140 Lys Ile Pro Leu Val Ile Ala Asn Ala Arg Leu Ser Ala Arg Ser Ala 145 150 155 160 Ala Gly Tyr Ala Lys Leu Gly Lys Phe Val Arg Arg Leu Leu Arg Arg 165 170 175 Ile Thr Leu Ile Ala Ala Gln Asn Glu Glu Asp Gly Ala Arg Phe Val 180 185 190 Ala Leu Gly Ala Lys Asn Asn Gln Val Thr Val Thr Gly Ser Leu Lys 195 200 205 Phe Asp Ile Ser Val Thr Pro Gln Leu Ala Ala Lys Ala Val Thr Leu 210 215 220 Arg Arg Gln Trp Ala Pro His Arg Pro Val Trp Ile Ala Thr Ser Thr 225 230 235 240 His Glu Gly Glu Glu Ser Val Val Ile Ala Ala His Gln Ala Leu Leu 245 250 255 Gln Gln Phe Pro Asn Leu Leu Leu Ile Leu Val Pro Arg His Pro Glu 260 265 270 Arg Phe Pro Asp Ala Ile Asn Leu Val Arg Gln Ala Gly Leu Ser Tyr 275 280 285 Ile Thr Arg Ser Ser Gly Glu Val Pro Ser Thr Ser Thr Gln Val Val 290 295 300 Val Gly Asp Thr Met Gly Glu Leu Met Leu Leu Tyr Gly Ile Ala Asp 305 310 315 320 Leu Ala Phe Val Gly Gly Ser Leu Val Glu Arg Gly Gly His Asn Pro 325 330 335 Leu Glu Ala Ala Ala His Ala Ile Pro Val Leu Met Gly Pro His Thr 340 345 350 Phe Asn Phe Lys Asp Ile Cys Ala Arg Leu Glu Gln Ala Ser Gly Leu 355 360 365 Ile Thr Val Thr Asp Ala Thr Thr Leu Ala Lys Glu Val Ser Ser Leu 370 375 380 Leu Thr Asp Ala Asp Tyr Arg Ser Phe Tyr Gly Arg His Ala Val Glu 385 390 395 400 Val Leu Tyr Gln Asn Gln Gly Ala Leu Gln Arg Leu Leu Gln Leu Leu 405 410 415 Glu Pro Tyr Leu Pro Pro Lys Thr His 420 425 <210> 23 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Escherichia coli KdtA polypeptide <400> 23 atgctcgaat tgctttacac cgcccttctc taccttattc agccgctgat ctggatacgg 60 ctctgggtgc gcggacgtaa ggctccggcc tatcgaaaac gctggggtga acgttacggt 120 ttttaccgcc atccgctaaa accaggcggc attatgctgc actccgtctc cgtcggtgaa 180 actctggcgg caatcccgtt ggtgcgcgcg ctgcgtcatc gttatcctga tttaccgatt 240 accgtaacaa ccatgacgcc aaccggttcg gagcgcgtac aatcggcttt cgggaaggat 300 gttcagcacg tttatctgcc gtatgatctg cccgatgcac tcaaccgttt cctgaataaa 360 gtcgacccta aactggtgtt gattatggaa accgaactat ggcctaacct gattgcggcg 420 ctacataaac gtaaaattcc gctggtgatc gctaacgcgc gactctctgc ccgctcggcc 480 gcaggttatg ccaaactggg taaattcgtc cgtcgcttgc tgcgtcgtat tacgctgatt 540 gctgcgcaaa atgaagaaga tggtgcacgt tttgtggcgc tgggcgcaaa aaataatcag 600 gtgaccgtta ccggtagcct gaaattcgat atttctgtaa cgccgcagtt ggctgctaaa 660 gccgtgacgc tgcgccgcca gtgggcacca caccgcccgg tatggattgc caccagcact 720 cacgaaggcg aagagagtgt ggtgatcgcc gcacatcagg cattgttaca gcaattcccg 780 aatttattgc tcatcctggt accccgtcat ccggaacgct tcccggatgc gattaacctt 840 gtccgccagg ctggactaag ctatatcaca cgctcttcag gggaagtccc ctccaccagc 900 acgcaggttg tggttggcga tacgatgggc gagttgatgt tactgtatgg cattgccgat 960 ctcgcctttg ttggcggttc actggttgaa cgtggtgggc ataatccgct ggaagctgcc 1020 gcacacgcta ttccggtatt gatggggccg catactttta actttaaaga catttgcgcg 1080 cggctggagc aggcaagcgg gctgattacc gttaccgatg ccactacgct tgcaaaagag 1140 gtttcctctt tactcaccga cgccgattac cgtagtttct atggccgtca tgccgttgaa 1200 gtactgtatc aaaaccaggg cgcgctacag cgtctgcttc aactgctgga accttacctg 1260 ccaccgaaaa cgcattga 1278 <210> 24 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Forward primer amplifying kdtA::frt-kan-frt <400> 24 gctaaataca tagaatcccc agcacatcca taagtcagct atttactatg ctcgaattgc 60 gtgtaggctg gagctgcttc 80 <210> 25 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer amplifying kdtA::frt-kan-frt <400> 25 atcgatatga ccattggtaa tgggatcgaa agtacccgga taaatcgccc gtttttgcat 60 tgaatatcct ccttagttcc tattcc 86 <110> EUBIOLOGICS CO., LTD. Korea Institute of Science and Technology <120> Method for producing phosphoryl lipid A <130> PN132814KR <150> KR 10-2019-0106640 <151> 2019-08-29 <160> 25 <170> KopatentIn 2.0 <210> 1 <211> 306 <212> PRT <213> Artificial Sequence <220> <223> Escherichia coli LpxL polypeptide <400> 1 Met Thr Asn Leu Pro Lys Phe Ser Thr Ala Leu Leu His Pro Arg Tyr 1 5 10 15 Trp Leu Thr Trp Leu Gly Ile Gly Val Leu Trp Leu Val Val Gln Leu 20 25 30 Pro Tyr Pro Val Ile Tyr Arg Leu Gly Cys Gly Leu Gly Lys Leu Ala 35 40 45 Leu Arg Phe Met Lys Arg Arg Ala Lys Ile Val His Arg Asn Leu Glu 50 55 60 Leu Cys Phe Pro Glu Met Ser Glu Gln Glu Arg Arg Lys Met Val Val 65 70 75 80 Lys Asn Phe Glu Ser Val Gly Met Gly Leu Met Glu Thr Gly Met Ala 85 90 95 Trp Phe Trp Pro Asp Arg Arg Ile Ala Arg Trp Thr Glu Val Ile Gly 100 105 110 Met Glu His Ile Arg Asp Val Gln Ala Gln Lys Arg Gly Ile Leu Leu 115 120 125 Val Gly Ile His Phe Leu Thr Leu Glu Leu Gly Ala Arg Gln Phe Gly 130 135 140 Met Gln Glu Pro Gly Ile Gly Val Tyr Arg Pro Asn Asp Asn Pro Leu 145 150 155 160 Ile Asp Trp Leu Gln Thr Trp Gly Arg Leu Arg Ser Asn Lys Ser Met 165 170 175 Leu Asp Arg Lys Asp Leu Lys Gly Met Ile Lys Ala Leu Lys Lys Gly 180 185 190 Glu Val Val Trp Tyr Ala Pro Asp His Asp Tyr Gly Pro Arg Ser Ser 195 200 205 Val Phe Val Pro Leu Phe Ala Val Glu Gln Ala Ala Thr Thr Thr Gly 210 215 220 Thr Trp Met Leu Ala Arg Met Ser Gly Ala Cys Leu Val Pro Phe Val 225 230 235 240 Pro Arg Arg Lys Pro Asp Gly Lys Gly Tyr Gln Leu Ile Met Leu Pro 245 250 255 Pro Glu Cys Ser Pro Pro Leu Asp Asp Ala Glu Thr Thr Ala Ala Trp 260 265 270 Met Asn Lys Val Val Glu Lys Cys Ile Met Met Ala Pro Glu Gln Tyr 275 280 285 Met Trp Leu His Arg Arg Phe Lys Thr Arg Pro Glu Gly Val Pro Ser 290 295 300 Arg Tyr 305 <210> 2 <211> 921 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Escherichia coli LpxL polypeptide <400> 2 atgacgaatc tacccaagtt ctccaccgca ctgcttcatc cgcgttattg gttaacctgg 60 ttgggtattg gcgtactttg gttagtcgtg caattgccct acccggttat ctaccgcctc 120 ggttgtggat taggaaaact ggcgttacgt tttatgaaac gacgcgcaaa aattgtgcat 180 cgcaacctgg aactgtgctt cccggaaatg agcgaacaag aacgccgtaa aatggtggtg 240 aagaatttcg aatccgttgg catgggcctg atggaaaccg gcatggcgtg gttctggccg 300 gaccgccgaa tcgcccgctg gacggaagtg atcggcatgg aacacattcg tgacgtgcag 360 gcgcaaaaac gcggcatcct gttagttggc atccattttc tgacactgga gctgggtgcg 420 cggcagtttg gtatgcagga accgggtatt ggcgtttatc gcccgaacga taatccactg 480 attgactggc tacaaacctg gggccgtttg cgctcaaata aatcgatgct cgaccgcaaa 540 gatttaaaag gcatgattaa agccctgaaa aaaggcgaag tggtctggta cgcaccggat 600 catgattacg gcccgcgctc aagcgttttc gtcccgttgt ttgccgttga gcaggctgcg 660 accacgaccg gaacctggat gctggcacgg atgtccggcg catgtctggt gcccttcgtt 720 ccacgccgta agccagatgg caaagggtat caattgatta tgctgccgcc agagtgttct 780 ccgccactgg atgatgccga aactaccgcc gcgtggatga acaaagtggt cgaaaaatgc 840 atcatgatgg caccagagca gtatatgtgg ttacaccgtc gctttaaaac acgcccggaa 900 ggcgttcctt cacgctatta a 921 <210> 3 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> LpxL forward primer P1 <400> 3 cgcagtctag aaaggagata tattgatgac gaatctaccc aagttctc 48 <210> 4 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> LpxL reverse primer P2 <400> 4 cgctattatt ttttttcgtt tccattggta tatctccttc ttattaatag cgtgaaggaa 60 cgccttc 67 <210> 5 <211> 323 <212> PRT <213> Artificial Sequence <220> <223> Escherichia coli LpxM polypeptide <400> 5 Met Glu Thr Lys Lys Asn Asn Ser Glu Tyr Ile Pro Glu Phe Asp Lys 1 5 10 15 Ser Phe Arg His Pro Arg Tyr Trp Gly Ala Trp Leu Gly Val Ala Ala 20 25 30 Met Ala Gly Ile Ala Leu Thr Pro Pro Lys Phe Arg Asp Pro Ile Leu 35 40 45 Ala Arg Leu Gly Arg Phe Ala Gly Arg Leu Gly Lys Ser Ser Arg Arg 50 55 60 Arg Ala Leu Ile Asn Leu Ser Leu Cys Phe Pro Glu Arg Ser Glu Ala 65 70 75 80 Glu Arg Glu Ala Ile Val Asp Glu Met Phe Ala Thr Ala Pro Gln Ala 85 90 95 Met Ala Met Met Ala Glu Leu Ala Ile Arg Gly Pro Glu Lys Ile Gln 100 105 110 Pro Arg Val Asp Trp Gln Gly Leu Glu Ile Ile Glu Glu Met Arg Arg 115 120 125 Asn Asn Glu Lys Val Ile Phe Leu Val Pro His Gly Trp Ala Val Asp 130 135 140 Ile Pro Ala Met Leu Met Ala Ser Gln Gly Gln Lys Met Ala Ala Met 145 150 155 160 Phe His Asn Gln Gly Asn Pro Val Phe Asp Tyr Val Trp Asn Thr Val 165 170 175 Arg Arg Arg Phe Gly Gly Arg Leu His Ala Arg Asn Asp Gly Ile Lys 180 185 190 Pro Phe Ile Gln Ser Val Arg Gln Gly Tyr Trp Gly Tyr Tyr Leu Pro 195 200 205 Asp Gln Asp His Gly Pro Glu His Ser Glu Phe Val Asp Phe Phe Ala 210 215 220 Thr Tyr Lys Ala Thr Leu Pro Ala Ile Gly Arg Leu Met Lys Val Cys 225 230 235 240 Arg Ala Arg Val Val Pro Leu Phe Pro Ile Tyr Asp Gly Lys Thr His 245 250 255 Arg Leu Thr Ile Gln Val Arg Pro Pro Met Asp Asp Leu Leu Glu Ala 260 265 270 Asp Asp His Thr Ile Ala Arg Arg Met Asn Glu Glu Val Glu Ile Phe 275 280 285 Val Gly Pro Arg Pro Glu Gln Tyr Thr Trp Ile Leu Lys Leu Leu Lys 290 295 300 Thr Arg Lys Pro Gly Glu Ile Gln Pro Tyr Lys Arg Lys Asp Leu Tyr 305 310 315 320 Pro Ile Lys <210> 6 <211> 972 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Escherichia coli LpxM polypeptide <400> 6 atggaaacga aaaaaaataa tagcgaatac attcctgagt ttgataaatc ctttcgccac 60 ccgcgctact ggggagcatg gctgggcgta gcagcgatgg cgggtatcgc tttaacgccg 120 ccaaagttcc gtgatcccat tctggcacgg ctgggacgtt ttgccggacg actgggaaaa 180 agctcacgcc gtcgtgcgtt aatcaatctg tcgctctgct ttccagaacg tagtgaagct 240 gaacgcgaag cgattgttga tgagatgttt gccaccgcgc cgcaagcgat ggcaatgatg 300 gctgagttgg caatacgcgg gccggagaaa attcagccgc gcgttgactg gcaagggctg 360 gagatcatcg aagagatgcg gcgtaataac gagaaagtta tctttctggt gccgcacggt 420 tgggccgtcg atattcctgc catgctgatg gcctcgcaag ggcagaaaat ggcagcgatg 480 ttccataatc agggcaaccc ggtttttgat tatgtctgga acacggtgcg tcgtcgcttt 540 ggcggtcgtc tgcatgcgag aaatgacggt attaaaccat tcatccagtc ggtacgtcag 600 gggtactggg gatattattt acccgatcag gatcatggcc cagagcacag cgaatttgtg 660 gatttctttg ccacctataa agcgacgttg cccgcgattg gtcgtttgat gaaagtgtgc 720 cgtgcgcgcg ttgtaccgct gtttccgatt tatgatggca agacgcatcg tctgacgatt 780 caggtgcgcc caccgatgga tgatctgtta gaggcggatg atcatacgat tgcgcggcgg 840 atgaatgaag aagtcgagat ttttgttggt ccgcgaccag aacaatacac ctggatacta 900 aaattgctga aaactcgcaa accgggcgaa atccagccgt ataagcgcaa agatctttat 960 cccatcaaat aa 972 <210> 7 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> LpxM forward primer P3 <400> 7 gaaggcgttc cttcacgcta ttaataagaa ggagatatac caatggaaac gaaaaaaaat 60 aatagcg 67 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> LpxM reverse primer P3 <400> 8 gcagaagctt ttatttgatg ggataaagat ctttgcg 37 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PL promoter nucleotide sequence <400> 9 ttgacataaa taccactggc ggtgatact 29 <210> 10 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Forward primer amplifying PL promoter <400> 10 ggcagtgagc gcaacgcaga attcttgaca taaataccac tggcggtgat actttcacac 60 aggaaacagc tatgacc 77 <210> 11 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer amplifying PL promoter <400> 11 ggtcatagct gtttcctgtg tgaaagtatc accgccagtg gtatttatgt caagaattct 60 gcgttgcgct cactgcc 77 <210> 12 <211> 190 <212> PRT <213> Artificial Sequence <220> <223> Helicobactor pylori LpxE mutant <400> 12 Met Lys Lys Phe Leu Phe Lys Gln Lys Phe Cys Glu Ser Leu Pro Lys 1 5 10 15 Ser Phe Ser Lys Thr Leu Leu Ala Leu Ser Leu Gly Leu Ile Leu Leu 20 25 30 Gly Ile Phe Ala Pro Phe Pro Lys Val Pro Lys Gln Pro Ser Val Pro 35 40 45 Leu Met Phe His Phe Thr Glu His Tyr Ala Arg Phe Ile Pro Thr Ile 50 55 60 Leu Ser Val Ala Ile Pro Leu Ile Gln Arg Asp Ala Val Gly Leu Phe 65 70 75 80 Gln Val Ala Asn Ala Ser Ile Ala Thr Thr Leu Leu Thr His Thr Thr 85 90 95 Lys Arg Ala Leu Asn His Val Thr Ile Asn Asp Gln Arg Leu Gly Glu 100 105 110 Arg Pro Tyr Gly Gly Asn Phe Asn Met Pro Ser Gly His Ser Ser Met 115 120 125 Val Gly Leu Ala Val Ala Phe Leu Met Arg Arg Tyr Ser Phe Lys Lys 130 135 140 Tyr Phe Trp Leu Leu Pro Leu Val Pro Leu Thr Met Leu Ala Arg Ile 145 150 155 160 Tyr Leu Asp Met His Thr Ile Gly Ala Val Leu Thr Gly Leu Gly Val 165 170 175 Gly Met Leu Cys Val Ser Leu Phe Thr Ser Pro Lys Lys Pro 180 185 190 <210> 13 <211> 573 <212> DNA <213> Artificial Sequence <220> <223> Polynucleodide encoding Helicobactor pylori LpxE mutant <400> 13 atgaaaaaat tcttatttaa acaaaaattt tgtgaaagcc tgcccaaatc gttttctaaa 60 actttgttag cgctcagttt gggcttgatt ttattaggca tttttgcgcc tttccctaaa 120 gtccctaaac agcctagcgt gcctttaatg tttcatttca ccgagcatta tgcgcgcttt 180 atccctacga ttttatctgt ggcgattccc ttaatccaaa gagatgcggt agggcttttt 240 caagtcgcta acgcttctat cgctacaacc cttctcacgc acaccaccaa aagagcctta 300 aaccatgtaa caatcaacga tcagcgtttg ggcgagcgcc cttatggagg taatttcaac 360 atgccaagcg ggcattcgtc tatggtgggt ttggcggtgg cgtttttaat gcgccgctat 420 tcttttaaaa aatacttttg gctcttgccc ctagtccctt tgaccatgct cgctcgcatt 480 tatttagaca tgcacaccat tggcgcggtg ctgaccgggc ttggcgttgg aatgttgtgc 540 gtaagccttt ttacaagccc caaaaagcct taa 573 <210> 14 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying HpLpxE mutant <400> 14 gatcctctag aaaggagata tattgatgaa aaaattctta tttaaacaaa aattt 55 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying Helicobactor pylori LpxE mutant <400> 15 agctacaagc ttttaaggct ttttggggc 29 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying frt-kan-frt <400> 16 gcagaagctt gtgtaggctg gagctgcttc 30 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying frt-kan-frt <400> 17 gcagaagctt atgaatatcc tccttagttc ctat 34 <210> 18 <211> 237 <212> PRT <213> Artificial Sequence <220> <223> Escherichia coli LpxT polypeptide <400> 18 Met Ile Lys Asn Leu Pro Gln Ile Val Leu Leu Asn Ile Val Gly Leu 1 5 10 15 Ala Leu Phe Leu Ser Trp Tyr Ile Pro Val Asn His Gly Phe Trp Leu 20 25 30 Pro Ile Asp Ala Asp Ile Phe Tyr Phe Phe Asn Gln Lys Leu Val Glu 35 40 45 Ser Lys Ala Phe Leu Trp Leu Val Ala Leu Thr Asn Asn Arg Ala Phe 50 55 60 Asp Gly Cys Ser Leu Leu Ala Met Gly Met Leu Met Leu Ser Phe Trp 65 70 75 80 Leu Lys Glu Asn Ala Pro Gly Arg Arg Arg Ile Val Ile Ile Gly Leu 85 90 95 Val Met Leu Leu Thr Ala Val Val Leu Asn Gln Leu Gly Gln Ala Leu 100 105 110 Ile Pro Val Lys Arg Ala Ser Pro Thr Leu Thr Phe Thr Asp Ile Asn 115 120 125 Arg Val Ser Glu Leu Leu Ser Val Pro Thr Lys Asp Ala Ser Arg Asp 130 135 140 Ser Phe Pro Gly Asp His Gly Met Met Leu Leu Ile Phe Ser Ala Phe 145 150 155 160 Met Trp Arg Tyr Phe Gly Lys Val Ala Gly Leu Ile Ala Leu Ile Ile 165 170 175 Phe Val Val Phe Ala Phe Pro Arg Val Met Ile Gly Ala His Trp Phe 180 185 190 Thr Asp Ile Ile Val Gly Ser Met Thr Val Ile Leu Ile Gly Leu Pro 195 200 205 Trp Val Leu Leu Thr Pro Leu Ser Asp Arg Leu Ile Thr Phe Phe Asp 210 215 220 Lys Ser Leu Pro Gly Lys Asn Lys His Phe Gln Asn Lys 225 230 235 <210> 19 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Escherichia coli LpxT polypeptide <400> 19 atgattaaaa atttgccgca aatagtgttg ttgaatattg tcggcctcgc gctgtttctt 60 tcctggtata tccccgttaa tcatggattc tggttgccga ttgatgcgga tattttttat 120 ttctttaatc agaaactggt cgaaagtaag gcctttttgt ggctggttgc attgaccaac 180 aatcgcgcct tcgacggttg ttcactgctg gcgatgggta tgttgatgct gagtttctgg 240 ctgaaagaaa acgcccctgg cagacgacgt atcgtgatta ttggtctggt catgctatta 300 actgcagtgg tattaaacca gctgggtcag gcattaattc ctgtaaaacg ggccagccca 360 acattgactt ttaccgatat taaccgcgtc agcgaactgc tctctgttcc cacgaaagat 420 gcctcacgag atagcctcc cggcgatcac ggcatgatgc tgcttatttt ttcggcattc 480 atgtggcgtt atttcggcaa agttgcaggc cttatcgccc ttattatttt tgtggttttt 540 gcatttccca gagtaatgat tggcgcacac tggtttactg acatcattgt cggttcgatg 600 accgtgatat tgatcggttt gccctgggtg ttgctgacgc cattaagtga tcgattaatc 660 accttttttg acaaatcact accaggaaaa aacaaacatt tccaaaacaa ataa 714 <210> 20 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Forward primer amplifying bacA::HpLpxE-frt-kan-frt <400> 20 aacctggtca tacgcagtag ttcggacaag cggtacattt taataattta ggggtttatt 60 gatgaaaaaa ttcttattta aacaaaaat 89 <210> 21 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer amplifying bacA::HpLpxE-frt-kan-frt <400> 21 tgacaacgcc aagcatccga cactattcct caattaaaag aacacgacat acaccgcagc 60 cgccacatga atatcctcct tagttccta 89 <210> 22 <211> 425 <212> PRT <213> Artificial Sequence <220> <223> Escherichia coli KdtA polypeptide <400> 22 Met Leu Glu Leu Leu Tyr Thr Ala Leu Leu Tyr Leu Ile Gln Pro Leu 1 5 10 15 Ile Trp Ile Arg Leu Trp Val Arg Gly Arg Lys Ala Pro Ala Tyr Arg 20 25 30 Lys Arg Trp Gly Glu Arg Tyr Gly Phe Tyr Arg His Pro Leu Lys Pro 35 40 45 Gly Gly Ile Met Leu His Ser Val Ser Val Gly Glu Thr Leu Ala Ala 50 55 60 Ile Pro Leu Val Arg Ala Leu Arg His Arg Tyr Pro Asp Leu Pro Ile 65 70 75 80 Thr Val Thr Thr Met Thr Pro Thr Gly Ser Glu Arg Val Gln Ser Ala 85 90 95 Phe Gly Lys Asp Val Gln His Val Tyr Leu Pro Tyr Asp Leu Pro Asp 100 105 110 Ala Leu Asn Arg Phe Leu Asn Lys Val Asp Pro Lys Leu Val Leu Ile 115 120 125 Met Glu Thr Glu Leu Trp Pro Asn Leu Ile Ala Ala Leu His Lys Arg 130 135 140 Lys Ile Pro Leu Val Ile Ala Asn Ala Arg Leu Ser Ala Arg Ser Ala 145 150 155 160 Ala Gly Tyr Ala Lys Leu Gly Lys Phe Val Arg Arg Leu Leu Arg Arg 165 170 175 Ile Thr Leu Ile Ala Ala Gln Asn Glu Glu Asp Gly Ala Arg Phe Val 180 185 190 Ala Leu Gly Ala Lys Asn Asn Gln Val Thr Val Thr Gly Ser Leu Lys 195 200 205 Phe Asp Ile Ser Val Thr Pro Gln Leu Ala Ala Lys Ala Val Thr Leu 210 215 220 Arg Arg Gln Trp Ala Pro His Arg Pro Val Trp Ile Ala Thr Ser Thr 225 230 235 240 His Glu Gly Glu Glu Ser Val Val Ile Ala Ala His Gln Ala Leu Leu 245 250 255 Gln Gln Phe Pro Asn Leu Leu Leu Ile Leu Val Pro Arg His Pro Glu 260 265 270 Arg Phe Pro Asp Ala Ile Asn Leu Val Arg Gln Ala Gly Leu Ser Tyr 275 280 285 Ile Thr Arg Ser Ser Gly Glu Val Pro Ser Thr Ser Thr Gln Val Val 290 295 300 Val Gly Asp Thr Met Gly Glu Leu Met Leu Leu Tyr Gly Ile Ala Asp 305 310 315 320 Leu Ala Phe Val Gly Gly Ser Leu Val Glu Arg Gly Gly His Asn Pro 325 330 335 Leu Glu Ala Ala Ala His Ala Ile Pro Val Leu Met Gly Pro His Thr 340 345 350 Phe Asn Phe Lys Asp Ile Cys Ala Arg Leu Glu Gln Ala Ser Gly Leu 355 360 365 Ile Thr Val Thr Asp Ala Thr Thr Leu Ala Lys Glu Val Ser Ser Leu 370 375 380 Leu Thr Asp Ala Asp Tyr Arg Ser Phe Tyr Gly Arg His Ala Val Glu 385 390 395 400 Val Leu Tyr Gln Asn Gln Gly Ala Leu Gln Arg Leu Leu Gln Leu Leu 405 410 415 Glu Pro Tyr Leu Pro Pro Lys Thr His 420 425 <210> 23 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Escherichia coli KdtA polypeptide <400> 23 atgctcgaat tgctttacac cgcccttctc taccttattc agccgctgat ctggatacgg 60 ctctgggtgc gcggacgtaa ggctccggcc tatcgaaaac gctggggtga acgttacggt 120 ttttaccgcc atccgctaaa accaggcggc attatgctgc actccgtctc cgtcggtgaa 180 actctggcgg caatcccgtt ggtgcgcgcg ctgcgtcatc gttatcctga tttaccgatt 240 accgtaacaa ccatgacgcc aaccggttcg gagcgcgtac aatcggcttt cgggaaggat 300 gttcagcacg tttatctgcc gtatgatctg cccgatgcac tcaaccgttt cctgaataaa 360 gtcgacccta aactggtgtt gattatggaa accgaactat ggcctaacct gattgcggcg 420 ctacataaac gtaaaattcc gctggtgatc gctaacgcgc gactctctgc ccgctcggcc 480 gcaggttatg ccaaactggg taaattcgtc cgtcgcttgc tgcgtcgtat tacgctgatt 540 gctgcgcaaa atgaagaaga tggtgcacgt tttgtggcgc tgggcgcaaa aaataatcag 600 gtgaccgtta ccggtagcct gaaattcgat atttctgtaa cgccgcagtt ggctgctaaa 660 gccgtgacgc tgcgccgcca gtgggcacca caccgcccgg tatggattgc caccagcact 720 cacgaaggcg aagagagtgt ggtgatcgcc gcacatcagg cattgttaca gcaattcccg 780 aatttattgc tcatcctggt accccgtcat ccggaacgct tcccggatgc gattaacctt 840 gtccgccagg ctggactaag ctatatcaca cgctcttcag gggaagtccc ctccaccagc 900 acgcaggttg tggttggcga tacgatgggc gagttgatgt tactgtatgg cattgccgat 960 ctcgcctttg ttggcggttc actggttgaa cgtggtgggc ataatccgct ggaagctgcc 1020 gcacacgcta ttccggtatt gatggggccg catactttta actttaaaga catttgcgcg 1080 cggctggagc aggcaagcgg gctgattacc gttaccgatg ccactacgct tgcaaaagag 1140 gtttcctctt tactcaccga cgccgattac cgtagtttct atggccgtca tgccgttgaa 1200 gtactgtatc aaaaccaggg cgcgctacag cgtctgcttc aactgctgga accttacctg 1260 ccaccgaaaa cgcattga 1278 <210> 24 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Forward primer amplifying kdtA::frt-kan-frt <400> 24 gctaaataca tagaatcccc agcacatcca taagtcagct atttactatg ctcgaattgc 60 gtgtaggctg gagctgcttc 80 <210> 25 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer amplifying kdtA::frt-kan-frt <400> 25 atcgatatga ccattggtaa tgggatcgaa agtacccgga taaatcgccc gtttttgcat 60 tgaatatcct ccttagttcc tattcc 86
Claims (18)
모노포스포릴 지질 A를 생산하는 세균을 배양하는 단계로서,
상기 세균은 생장곡선에서 지수기(exponential phase) 시작점으로부터 정지기(stationary phase) 시작점 후 5시간까지 배양되는 것인 단계;
배양물로부터 상기 세균을 수득하는 단계;
수득된 세균으로부터 지질을 수득하는 단계; 및
수득된 지질로부터 모노포스포릴 지질 A를 수득하는 단계를 포함하는 방법.As a method of producing monophosphoryl lipid A (MPLA), the method comprises:
As the step of culturing a bacterium producing monophosphoryl lipid A,
Wherein the bacterium is cultured from the starting point of the exponential phase to 5 hours after the starting point of the stationary phase in the growth curve;
Obtaining the bacteria from the culture;
Obtaining lipids from the obtained bacteria; And
A method comprising the step of obtaining monophosphoryl lipid A from the obtained lipid.
Priority Applications (2)
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US17/630,657 US20220259633A1 (en) | 2019-08-29 | 2020-08-26 | Method for producing monophosphoryl lipid a |
PCT/KR2020/011425 WO2021040414A1 (en) | 2019-08-29 | 2020-08-26 | Method for producing monophosphoryl lipid a |
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KR20190106640 | 2019-08-29 | ||
KR1020190106640 | 2019-08-29 |
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KR20210027132A true KR20210027132A (en) | 2021-03-10 |
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KR1020200107412A KR20210027132A (en) | 2019-08-29 | 2020-08-25 | Method for producing monophosphoryl lipid A |
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KR (1) | KR20210027132A (en) |
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2020
- 2020-08-25 KR KR1020200107412A patent/KR20210027132A/en not_active Application Discontinuation
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