KR101863892B1 - Mannosylphosphorylation Reaction Using Recombinant MNN14 Protein - Google Patents
Mannosylphosphorylation Reaction Using Recombinant MNN14 Protein Download PDFInfo
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Abstract
본 발명은 Mnn4 단백질 및 서열번호 1로 표시되는 단백질의 활성이 모두 내재적 활성에 비하여 약화된, 재조합 효모 균주 및 이를 이용하여 인간화된 당사슬이 부착된 재조합 당단백질을 제조하는 방법에 관한 것이다. The present invention relates to a recombinant yeast strain in which the activity of the Mnn4 protein and the protein of SEQ ID NO: 1 is weaker than the intrinsic activity, and a method for producing a recombinant glycoprotein having the humanized oligosaccharide by using the same.
Description
본 발명은 Mnn4 단백질 및 Mnn14 단백질의 활성이 모두 내재적 활성에 비하여 약화된, 재조합 효모 균주 및 이를 이용하여 인간화된 당사슬이 부착된 재조합 당단백질을 제조하는 방법에 관한 것이다.The present invention relates to a recombinant yeast strain in which the activities of both the Mnn4 protein and the Mnn14 protein are weakened compared to the intrinsic activity, and a method for producing a recombinant glycoprotein to which a humanized sugar chain is attached using the same.
당질화를 통해서 당사슬이 부가되는 당단백질들은 현재 전세계 재조합 단백질 의약품 시장의 60% 이상을 차지하며 시장을 주도하고 있다. 특히, 당사슬은 당단백질 의약품의 치료 효능, 체내 지속성, 타겟팅 및 면역반응 등에서 중요한 역할을 하기에 의약품의 품질을 결정하는 주요 인자로 부각되고 있다. 당사슬이 부가되는 반응은 크게 N-결합 또는 O-결합 당질화(N-linked or O-linked glycosylation)의 두 가지 형태가 있으며, 이 중 N-결합 당질화를 통해서 부가되는 당사슬을 N-당사슬(N-glycan)이라 부르며 소포체에서 시작된다. 먼저 소포체 막에 존재하는 돌리콜 피로인산(dolichol pyrophosphate, PP-Dol)에 연결된 형태의 당사슬인 Glc3Man9GlcNAc2-PP-Dol을 형성한다. 그리고 올리고당 전이효소(oligosaccharyltransferase)가 이를 co-translational translocation 기작으로 리보좀에서 소포체로 번역되어 나오는 단백질 중에서 N-X-S/T 서열의 N-당질화 시퀀(sequon)에 전달한다. 소포체 안에 존재하는 당단백질 폴딩의 품질 제어를 담당하는 기작의 글루코시다아제(glucosidase)와 만노시다제(mannosidase)에 의해서 말단에 존재하는 포도당과 특정 만노스(mannose)가 제거되어 Man8GlcNAc2 구조를 갖는 고-만노스 형(high-mannose type) 당사슬을 부착한 상태로 골지체에 옮겨지게 된다. Glycoproteins to which sugar chains are added through glycosylation currently lead the market, accounting for more than 60% of the global recombinant protein pharmaceutical market. In particular, sugar chains are emerging as a major factor in determining the quality of drugs because they play an important role in the therapeutic efficacy, persistence in the body, targeting and immune response of glycoprotein drugs. The addition reaction in which sugar chain is largely N - O bond or a - combination glycosylation and two types of (N -linked or O -linked glycosylation) , the of the N - a sugar chain which is added via the binding glycosylation N - oligosaccharide ( N- glycan) and starts in the endoplasmic reticulum. First, it forms Glc 3 Man 9 GlcNAc 2 -PP-Dol, a sugar chain in the form of linked to dolychol pyrophosphate (PP-Dol) present in the endoplasmic reticulum membrane. And oligosaccharide transferase (oligosaccharyltransferase) transfers this to the N -glycosylation sequence (sequon) of the NXS/T sequence among proteins that are translated from the ribosome to the endoplasmic reticulum through a co-translational translocation mechanism. Glucosidase and mannosidase, which are the mechanisms responsible for controlling the quality of glycoprotein folding in the endoplasmic reticulum, remove glucose and specific mannose at the end, resulting in the Man 8 GlcNAc 2 structure. It is transferred to the Golgi body with the attached high-mannose type sugar chain.
상기에서 소개한 소포체에서 이루어지는 N-당사슬의 초기 생합성 과정은 진핵 미생물인 효모에서 고등동물인 포유류에 이르기까지 거의 동일한 과정으로 보존되어 있다. 그러나, 골지체로 넘어간 당사슬은 각 종에 특이적으로 다양한 당사슬 수식 과정을 거치게 되며, 그 결과로 효모, 곤충, 식물 및 동물 등에서 완전히 다른 형태의 당사슬들이 만들어지게 된다. 고등 동물에서는 골지체로 들어온 당단백질의 당사슬들은 만노시다제들에 의해서 Man5GlcNAc2 형태로 다듬어진다. 그리고 여기에 N-acetylglucosaminyltransferase (GNT) I이 작용해서 GlcNAc이 하나 부가된 후, 만노시다제 Ⅱ가 작용해서 반대편 가지에 부가되어 있던 만노스 2개를 더 제거하여 trimannosyl core (Man3GlcNAc2) 당사슬에 GlcNAc이 하나 부가되어 있는 혼합형 구조가 만들어진다. 이후 만노스가 제거된 가지에 GNT Ⅱ가 작용해서 GlcNAc이 하나 더 부가되면서 두 개의 안테나 구조를 갖는 당사슬이 생성된다. 이후에 GNT Ⅳ와 Ⅴ 등이 작용해서 네 개의 안테나 구조가 만들어지기도 하며, 일부에서는 GNT Ⅵ, Ⅸ 또는 VB 등이 작용해서 6개의 안테나 구조까지 만들어지는 경우도 있다. GlcNAc이 부가되어 안테나의 골격이 만들어진 후에 골지체에 존재하는 베타-갈락토실트랜스퍼라아제와 알파-시알산트랜스퍼라아제(alpha-sialyltrasnferase) 등이 작용해서 GlcNAc 위에 갈락토즈와 시알산이 부가된 복합형 당사슬 구조가 만들어진다. The initial biosynthesis process of the N -sugar chain in the endoplasmic reticulum introduced above has been preserved in almost the same process from yeast as eukaryotic microorganisms to mammals as higher animals. However, the sugar chains passed to the Golgi body undergo various sugar chain modification processes specifically for each species, and as a result, completely different types of sugar chains are made in yeast, insects, plants and animals. In higher animals, the sugar chains of glycoproteins that have entered the Golgi body are trimmed into Man 5 GlcNAc 2 form by mannosidase. In addition, after N- acetylglucosaminyltransferase (GNT) I acts to add one GlcNAc, mannosidase Ⅱ acts to remove two more mannose added to the opposite branch, and the trimannosyl core (Man 3 GlcNAc 2 ) is added to the sugar chain. A hybrid structure with one GlcNAc added is created. After that, GNT II acts on the branch from which mannose has been removed, and one more GlcNAc is added to create a sugar chain with two antenna structures. After that, GNT IV and V work together to create four antenna structures, and in some cases, GNT VI, IX, or VB works to create up to six antenna structures. After GlcNAc is added to create the skeleton of the antenna, the beta-galactosyltransferase and alpha-sialyltrasnferase present in the Golgi body act, and galactose and sialic acid are added on the GlcNAc. A sugar chain structure is created.
효모는 진핵 미생물로서 유전자 조작이 용이하여 다루기 쉽고 저렴한 비용으로 대량의 단백질을 생산할 수 있어서 경제성이 높을 뿐만 아니라, 인간을 감염시키는 바이러스와 프라이온 등에 오염이 될 가능성이 없는 등 안전성 또한 매우 높다는 많은 장점들을 가지고 있다. 그러나, 효모에서 합성된 당사슬 구조는 인간의 것과 매우 상이하여 인체 주입 시 면역 반응을 일으키는 문제점이 있다. 효모는 소포체까지는 고등동물과 동일한 당사슬 생합성 과정을 가지고 있으나, 골지체(Golgi)로 이동한 후에는 만노스가 추가로 부가되는 효모 특이적인 당사슬 수식 경로를 갖는다. 즉, OCH1 유전자 산물에 의해서 소포체에서 넘어온 Man8GlcNAc2 당사슬에 α(1,6)-결합으로 만노스가 부가되는 당사슬 외쇄(glycan outer chain) 개시 반응이 일어나며, 이를 시작으로 α(1,6)-결합 및 α(1,2)-결합으로 만노스가 연속적으로 부가되어 당사슬 외쇄가 합성된다. 전통효모인 사카로마이세스 세레비지애(Saccharomyces cerevisiae)의 경우에는 핵심 당사슬에 50-200개의 만노스가 연속적으로 부가되는 과당화 반응이 일어나기도 하며, 인체에서 항원으로 인지될 수 있는 α(1,3)-만노스가 MNN1 유전자 산물에 의해서 부가된다. 또한, 당사슬에 만노스-1-인산이 추가로 부가되는 만노스인산화(mannosylphosphorylation)를 통해서 "만노스-1-인산-6-O-만노스(mannose-1-phosphate-6-O-mannose)" 형태의 산성 당사슬이 생성된다는 사실도 알려졌다. 이는 주로 MNN6 유전자가 발현하는 효소에 의해서 만노스인산화 반응에 의해서 일어나며, 이를 제어하는 단백질을 발현하는 유전자로 MNN4가 제시된 바 있다(Odani et al., Glycobiol., 6:805, 1996; Odani et al., FEBS Lett., 420:1860, 1997). Yeast is a eukaryotic microorganism, which is easy to manipulate, so it is easy to handle and can produce a large amount of protein at low cost, so it is economical, and there are many advantages of high safety, such as no possibility of contamination such as viruses and prions that infect humans. Have them. However, the structure of the sugar chain synthesized from yeast is very different from that of humans, and thus there is a problem in causing an immune response when injected into the human body. Yeast has the same sugar chain biosynthesis process as in higher animals up to the endoplasmic reticulum, but has a yeast-specific sugar chain modification pathway in which mannose is additionally added after moving to the Golgi body. That is, the initiation reaction of the glycan outer chain in which mannose is added by α(1,6)-linkage to the Man 8 GlcNAc 2 sugar chain transferred from the endoplasmic reticulum by the OCH1 gene product occurs, starting with α(1,6) Mannose is continuously added by -bond and α(1,2)-bond to synthesize the outer chain of the sugar chain. In the case of Saccharomyces cerevisiae , a traditional yeast, a fructose reaction occurs in which 50-200 mannose is successively added to the core sugar chain, and α(1, 3) -mannose is added by the MNN1 gene product. Also, mannose-1-phosphate to a sugar chain is added by the mannose phosphorylation (mannosylphosphorylation) is added to "mannose-1-phosphate -6- O - mannose (mannose-1-phosphate-6- O -mannose)" in the form of acid It is also known that sugar chains are formed. This is mainly caused by mannose phosphorylation by an enzyme expressed by the MNN6 gene, and MNN4 has been suggested as a gene expressing a protein that controls it (Odani et al., Glycobiol., 6:805, 1996; Odani et al. , FEBS Lett., 420: 1860, 1997).
상기에서 기술한 바와 같이 효모에서 생산한 당단백질에 부착된 당사슬은 인간의 것과 구조가 달라서 인체 주입 시 면역 반응을 일으키기 때문에 의약용으로 사용할 수 없으므로, 이러한 문제를 해결하기 위해서 효모 특이적인 당사슬을 부가하는 당전이효소 유전자들을 파쇄하는 방법들이 제시되었다. 특히 S. cerevisiae 효모에서는 당사슬 외쇄 연장 반응을 개시하는 OCH1 유전자와 α(1,3)-만노스를 부가하는 MNN1 유전자 등을 파쇄하여 효모 특이적인 당사슬의 부가를 막는 방법들이 제시되었다(Nakayama et al., EMBO J, 11:2511, 1992; Nakanishi-Shindo et al., J Biol Chem, 268:26338, 1993).As described above, the sugar chain attached to the glycoprotein produced from yeast has a different structure from that of humans, so it can not be used for medicine because it causes an immune response when injected into the human body.To solve this problem, a yeast-specific sugar chain is added. Methods for disrupting glycotransferase genes have been suggested. In particular, in S. cerevisiae yeast, OCH1 gene and α (1,3) - to break through the MNN1 gene, such as for adding mannose presented are methods to prevent the addition of yeast-specific sugar chain (Nakayama et al, EMBO J, 11: 2511, 1992; Nakanishi-Shindo. et al., J Biol Chem, 268:26338, 1993).
또한, 상기에서 언급한 MNN4 및 MNN6에 의해서 만노스인산이 부가되어 생기는 "만노스-1-인산-6-O-만노스" 형태의 당사슬 가지도 인체 주입 시 면역 반응을 일으킬 수 있으므로, 대부분의 의약용 당단백질 생산을 위해서는 이를 제거해 주어야 한다. 따라서 S. cerevisiae 효모에서 효모 특이적인 당사슬의 생합성을 막기 위해서 보통 OCH1과 MNN1 유전자와 함께 만노스인산의 부가를 제어한다고 알려진 MNN4 유전자도 함께 결손 한다(Chiba et al., J Biol Chem 273:26298, 1998). 그러나 MNN4 유전자의 결손이 만노스인산의 부가 활성을 완전히 제거하지 못한다(Odani et al., Glycobiol 6:805, 1996). 따라서 만노스인산의 부가 활성을 지니는 다른 유전자가 있다고 추정되었으나 어떤 유전자가 이러한 활성을 담당하는지는 아직까지 보고되지 않았다. In addition, sugar chain branches in the form of "mannose-1-phosphate-6- O -mannose" that are produced by the addition of mannose phosphate by MNN4 and MNN6 mentioned above can cause an immune reaction when injected into the human body. In order to produce protein, it must be removed. Therefore, even with defects MNN4 gene known to control the addition of mannose phosphate with normal OCH1 gene and MNN1 to prevent yeast-specific sugar chain biosynthesis in S. cerevisiae yeast (Chiba et al, J Biol Chem 273:. 26298, 1998 ). However, the deletion of the MNN4 gene does not completely eliminate the addition activity of mannose phosphate (Odani et al ., Glycobiol 6:805, 1996). Therefore, it was presumed that there were other genes with the additional activity of mannose phosphate, but it has not been reported yet which genes are responsible for this activity.
이에, 본 발명에서는 효모, 특히 S. cerevisiae에서 만노스 인산을 당사슬에 부가하는 활성을 완전히 제거하기 위한 기술을 개발하고자 예의 노력한 결과, S. cerevisiae 균주에서 Mnn14 유전자가 만노스 인산화에 관여하며, MNN4 유전자와 MNN14 유전자의 이중 결손 시에 만노스 인산의 부가가 제거되는 것을 규명하여, 본 발명을 완성하였다. Therefore, in the present invention, as a result of earnest efforts to develop a technology for completely removing the activity of adding mannose phosphate to sugar chains in yeast, particularly S. cerevisiae , the Mnn14 gene is involved in mannose phosphorylation in S. cerevisiae strains, and MNN4 The present invention was completed by finding out that the addition of mannose phosphate is eliminated when the gene and the MNN14 gene are double deleted.
본 발명의 하나의 목적은 만노스 인산의 부가가 제어된, 재조합 효모 균주를 제공하는 것이다.One object of the present invention is to provide a recombinant yeast strain in which the addition of mannose phosphate is controlled.
본 발명의 다른 목적은 상기 재조합 효모 균주를 이용하여 재조합 당단백질을 제조하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing a recombinant glycoprotein using the recombinant yeast strain.
본 발명의 또 다른 목적은 만노스 인산이 부가되지 않은 재조합 당단백질의 제조에 사용하기 위한 상기 재조합 효모 균주의 용도를 제공하는 것이다. Another object of the present invention is to provide the use of the recombinant yeast strain for use in the production of a recombinant glycoprotein to which mannose phosphate is not added.
본 발명의 또 다른 목적은 당단백질에서 만노스 인산의 부가를 제어하는 것에 사용하기 위한, 서열번호 1로 표시되는 단백질의 용도를 제공하는 것이다. Another object of the present invention is to provide the use of a protein represented by SEQ ID NO: 1 for use in controlling the addition of mannose phosphate in a glycoprotein.
본 발명의 또 다른 목적은 만노스인산화에 사용하기 위한, 서열번호 1로 표시되는 단백질의 용도를 제공하는 것이다. Another object of the present invention is to provide the use of the protein represented by SEQ ID NO: 1 for use in mannose phosphorylation.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.
본 발명은 하나의 양태로서, 만노스 인산의 부가가 제어된, 재조합 효모 균주를 제공한다. 구체적으로, 상기 효모 균주는 비자연적으로 발생된 것일 수 있으나, 이에 제한되는 것은 아니다.In one aspect, the present invention provides a recombinant yeast strain in which the addition of mannose phosphate is controlled. Specifically, the yeast strain may be non-naturally occurring, but is not limited thereto.
구체적으로, 본 발명은 Mnn4 단백질 및 Mnn14 단백질의 활성이 모두 내재적 활성에 비하여 약화된, 재조합 효모 균주를 제공한다. Specifically, the present invention provides a recombinant yeast strain in which the activities of both the Mnn4 protein and the Mnn14 protein are weakened compared to the intrinsic activity.
본 발명에서 용어, “Mnn4 단백질”은 당사슬(올리고당[oligosaccharide]이라고도 함)의 만노스인산화에 관여하는 단백질이다. 상기 Mnn4 단백질은 만노스인산 전이효소(mannosylphosphate transferase)의 추정상의 양성 조절자(putative positive regulator)로 알려져 있다. 또한, 상기 Mnn4 단백질은 YKL200C, YKL201C로도 명명된다. 상기 Mnn4 단백질 및 이를 코딩하는 유전자 정보는 미국 국립 보건원 GenBank와 같은 데이터베이스를 통하여 얻을 수 있으며, 그 예로 상기 Mnn4 단백질은 서열번호 3으로 표시되는 아미노산 서열 (서열번호 4의 염기 서열)을 가질 수 있으나, 이에 제한되지 않는다.In the present invention, the term "Mnn4 protein" refers to a protein involved in the mannose phosphorylation of sugar chains (also referred to as oligosaccharides). The Mnn4 protein is known as a putative positive regulator of mannosylphosphate transferase. In addition, the Mnn4 protein is also referred to as YKL200C and YKL201C. The Mnn4 protein and gene information encoding it can be obtained through a database such as GenBank of the National Institutes of Health, for example, the Mnn4 protein may have an amino acid sequence represented by SEQ ID NO: 3 (base sequence of SEQ ID NO: 4), It is not limited thereto.
또한, 상기 Mnn4 단백질은 서열번호 3으로 표시되는 아미노산 서열을 가지는 단백질뿐만 아니라, 서열번호 3과 80% 이상, 구체적으로는 90% 이상, 보다 구체적으로는 95% 이상, 보다 더욱 구체적으로는 99% 이상의 상동성을 가지는 단백질로서, 실질적으로 Mnn4 단백질과 동일하거나 상응하는 생물학적 활성을 가지는 아미노산 서열이라면, 일부 서열이 결실, 변형, 치환 또는 부가되는 아미노산 서열을 갖는 경우도 본 발명의 범주에 포함하며, 이는 당업자에게 자명하다. In addition, the Mnn4 protein is not only a protein having an amino acid sequence represented by SEQ ID NO: 3, but also SEQ ID NO: 3 and 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 99% As a protein having the above homology, if an amino acid sequence having a biological activity substantially the same as or corresponding to the Mnn4 protein, some sequences have an amino acid sequence to be deleted, modified, substituted, or added to the scope of the present invention, This is obvious to a person skilled in the art.
본 발명에서 사용된 용어, “상동성”은 두 개의 폴리뉴클레오티드 또는 폴리펩타이드 모이티 사이의 동일성의 퍼센트를 말한다. 하나의 모이티로부터 다른 하나의 모이티까지의 서열 간 상동성은 알려진 당해 기술에 의해 결정될 수 있다. 예를 들면, 상동성을 서열정보를 정렬하고 용이하게 입수 가능한 컴퓨터 프로그램을 이용하여 두 개의 폴리뉴클레오티드 분자 또는 두 개의 폴리펩타이드 분자 간의 서열 정보를 직접 정렬하여 결정될 수 있다. 상기 컴퓨터 프로그램은 BLAST(NCBI), CLC Main Workbench(CLC bio), MegAlignTM(DNASTAR Inc) 등일 수 있다. 또한, 폴리뉴클레오티드 간 상동성은 상동 영역 간의 안정된 이중가닥을 이루는 조건 하에서 폴리뉴클레오티드의 혼성화한 후, 단일-가닥-특이적 뉴클레아제로 분해시켜 분해된 단편의 크기를 결정함으로써 결정할 수 있다.As used herein, the term “homology” refers to the percent of identity between two polynucleotides or polypeptide moieties. Homology between sequences from one moiety to another moiety can be determined by known art techniques. For example, homology can be determined by aligning sequence information and directly aligning sequence information between two polynucleotide molecules or two polypeptide molecules using an easily available computer program. The computer program may be BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), or the like. In addition, homology between polynucleotides can be determined by hybridizing polynucleotides under conditions forming a stable double-stranded between homologous regions, and then determining the size of the decomposed fragment by digesting with a single-stranded-specific nuclease.
본 발명에서 용어, “Mnn14 단백질”은 S. cerevisiae에서 MNN4 유전자와 파라로그인 유전자에 의하여 인코딩되는 단백질을 의미한다. 상기 Mnn14는 YJR061W로도 명명된다. 또한, 본 발명에서는 상기 Mnn14를 Mnn4pa로도 명명하였으며, 본 명세서에서 상기 용어들은 서로 혼용되어 사용될 수 있다. 상기 Mnn14 단백질의 정보는 미국 국립 보건원 GenBank와 같은 공지된 데이터베이스를 통하여 얻을 수 있으며, 그 예로 NP_012595인 단백질(서열번호 1)일 수 있으나, 이에 제한되지 않는다. In the present invention, the term “Mnn14 protein” refers to a protein encoded by the MNN4 gene and the paralog gene in S. cerevisiae. The Mnn14 is also named YJR061W. In addition, in the present invention, the Mnn14 is also named Mnn4pa, and in the present specification, the terms may be used interchangeably with each other. The information on the Mnn14 protein can be obtained through a known database such as GenBank of the National Institutes of Health, and may be, for example, a protein of NP_012595 (SEQ ID NO: 1), but is not limited thereto.
또한, 본 발명에서 상기 서열번호 1로 표시되는 Mnn14 단백질의 범주에는 서열번호 1의 아미노산 서열뿐만 아니라, 서열번호 1과 80% 이상, 구체적으로는 90% 이상, 보다 구체적으로는 95% 이상, 보다 더욱 구체적으로는 99% 이상의 상동성을 가지는 단백질로서, 실질적으로 서열번호 1의 아미노산 서열을 가지는 단백질과 동일하거나 상응하는 생물학적 활성을 가지는 아미노산 서열이라면, 일부 서열이 결실, 변형, 치환 또는 부가되는 아미노산 서열을 갖는 경우도 본 발명의 범주에 포함하며, 이는 당업자에게 자명하다.In addition, in the category of the Mnn14 protein represented by SEQ ID NO: 1 in the present invention, not only the amino acid sequence of SEQ ID NO: 1, but also 80% or more, specifically 90% or more, more specifically 95% or more, more More specifically, as a protein having 99% or more homology, if an amino acid sequence having biological activity substantially identical to or corresponding to the protein having the amino acid sequence of SEQ ID NO: 1, some sequences are deleted, modified, substituted or added amino acids The case of having a sequence is also included in the scope of the present invention, which is obvious to those skilled in the art.
상기 서열번호 1의 아미노산 서열을 가지는 단백질은 서열번호 2로 표시되는 염기서열로 인코딩되는 것일 수 있으나, 이에 제한되지 않으며, 코돈의 축퇴성(codon degeneracy)으로 인하여 상기 단백질을 코딩하는 염기 서열이 다양할 수 있음은 당업자에게 자명하다. The protein having the amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto, and the base sequence encoding the protein varies due to codon degeneracy. It is obvious to a person skilled in the art that it can be done.
본 발명에서 용어, “내재적 활성에 비하여 약화된”은 상기 미생물이 천연의 상태에서 가지고 있는 단백질의 활성과 비교하였을 때, 그 활성이 감소되거나, 활성이 없는 것을 모두 포함하는 개념이다. In the present invention, the term "weakened compared to intrinsic activity" refers to a concept including all of the activity of the microorganism having decreased or no activity when compared to the activity of the protein possessed by the microorganism in its natural state.
상기 약화는 단백질의 활성이 약화되도록 변이된 것일 수 있고, 상기 변이는 비자연적으로 발생된 것일 수 있으나, 이에 제한되는 것은 아니다. 구체적으로, 본 발명에서 단백질의 활성을 약화시키는 것은 상기 단백질을 암호화하는 염색체상의 유전자의 전체 또는 일부를 결실시키는 방법; 상기 단백질의 활성이 감소되도록 돌연변이된 유전자로, 염색체상의 상기 단백질을 암호화하는 유전자를 대체하는 방법; 상기 단백질을 암호화하는 염색체상의 유전자의 발현 조절 서열에 변이를 도입하는 방법; 상기 단백질을 암호화하는 유전자의 발현 조절 서열을 활성이 약하거나 없는 서열로 교체하는 방법; 상기 염색체상의 유전자의 전사체에 상보적으로 결합하여 상기 mRNA로부터 단백질로의 번역을 저해하는 안티센스 올리고뉴클레오티드를 도입하는 방법; 상기 단백질을 암호화하는 유전자의 SD 서열 앞단에 SD 서열과 상보적인 서열을 인위적으로 부가하여 2차 구조물을 형성시켜 리보솜(ribosome)의 부착이 불가능하게 만드는 법 및 해당 서열의 ORF(open reading frame)의 3' 말단에 역전사되도록 프로모터를 부가하는 RTE(Reverse transcription engineering) 방법으로 이루어지는 군에서 선택되는 방법으로 수행될 수 있으나, 이에 제한되지 않으며, 단백질 활성을 약화시키는 방법이라면 어떠한 것이라도 적용될 수 있음은 당업자에게 자명하다. The attenuation may be mutated so that the activity of the protein is weakened, and the mutation may be non-naturally occurring, but is not limited thereto. Specifically, weakening the activity of a protein in the present invention is a method of deleting all or part of a gene on a chromosome encoding the protein; A method of replacing a gene encoding the protein on a chromosome with a gene mutated to decrease the activity of the protein; A method of introducing a mutation into an expression control sequence of a gene on a chromosome encoding the protein; A method of replacing the expression control sequence of the gene encoding the protein with a sequence having weak or no activity; A method of introducing an antisense oligonucleotide that inhibits translation from the mRNA into a protein by complementarily binding to the transcript of the gene on the chromosome; A method of making it impossible to attach a ribosome by artificially adding a sequence complementary to the SD sequence to the front end of the SD sequence of the gene encoding the protein to make it impossible to attach a ribosome, and of the ORF (open reading frame) of the sequence. It may be performed by a method selected from the group consisting of RTE (Reverse transcription engineering) method in which a promoter is added to reverse transcription at the 3'end, but is not limited thereto, and any method to attenuate protein activity may be applied. Self-explanatory to
또한, 상기 재조합 효모 균주는 Mnn1 단백질, Och1 단백질, 또는 둘 다의 활성이 내재적 활성에 비하여 약화된 것일 수 있다.In addition, the recombinant yeast strain may be one in which the activity of the Mnn1 protein, the Och1 protein, or both is weakened compared to the intrinsic activity.
상기 Mnn1 단백질은 알파-1,3-만노스전이효소(alpha-1,3-mannosyltransferase) 활성을 가진다. 상기 단백질은 인체에서 항원으로 인지될 수 있는 α(1,3)-만노스를 당단백질에 부가할 수 있으므로, 상기 단백질의 활성을 내재적 활성에 비하여 약화시킴으로써 재조합 당단백질에 효모 특이적 당사슬의 부가를 제어하는데 도움을 줄 수 있다. The Mnn1 protein has alpha-1,3-mannosyltransferase activity. Since the protein can add α(1,3)-mannose, which can be recognized as an antigen in the human body, to the glycoprotein, the activity of the protein is weakened compared to the intrinsic activity, thereby preventing the addition of yeast-specific sugar chains to the recombinant glycoprotein. It can help you control.
또한, Och1 단백질은 cis-골지체에서 만노스 전이효소로 작용하며, 당단백질의 N-연결된 당사슬의 폴리만노스 당 체인 연장(polymannose outer chain elongation)을 매개한다. 따라서, 상기 단백질의 활성을 내재적 활성에 비하여 약화시킴으로써 재조합 당단백질에 효모 특이적 당사슬의 부가를 제어하는데 도움을 줄 수 있다.In addition, the Och1 protein acts as a mannose transferase in the cis-Golgi body and mediates the polymannose outer chain elongation of the N -linked sugar chain of the glycoprotein. Therefore, by weakening the activity of the protein compared to the intrinsic activity, it can help to control the addition of yeast-specific sugar chains to the recombinant glycoprotein.
상기 기술된 Mnn1 단백질, Och1 단백질에 대한 정보는 상기 기술된 바와 같이 미국 국립 보건원 GenBank와 같은 당업자에게 공지된 데이터 베이스를 통하여 얻을 수 있다. Information on the Mnn1 protein and Och1 protein described above can be obtained through a database known to those skilled in the art, such as the National Institutes of Health GenBank, as described above.
또한, 상기 단백질의 활성 약화에 대해서도 앞서 설명한 내용이 적용된다. In addition, the above description applies to the weakening of the activity of the protein.
또한, 상기 효모는 사카로마이세스 세레비지애 (saccharomycens cerevisiae)일 수 있으나, 이에 제한되지 않으며, Mnn4 및 Mnn14 단백질의 활성 약화로 효모 특이적 당사슬 제어를 달성할 수 있는 효모라면 본 발명의 범주에 포함될 수 있다. In addition, the yeast is Saccharomycens cerevisiae ( saccharomycens cerevisiae ), but is not limited thereto, and any yeast capable of achieving yeast-specific sugar chain control by weakening the activity of Mnn4 and Mnn14 proteins may be included in the scope of the present invention.
상기 기술된 Mnn1, Och1, Mnn4 및 Mnn14 단백질들의 활성이 약화된, 효모 특이적 당사슬 생합성 경로가 약화된 효모가 발현하는 당단백질들에 부착된 N-당사슬들은 대부분이 Man8GlcNAc2 구조를 가질 수 있다. 당사슬 수식 효소 활성을 갖는 하나 이상의 단백질을 발현시킬 수 있는 발현 벡터로 상기한 변이주를 형질전환시키는 경우 더욱 효과적으로 인간의 당사슬과 유사한 구조를 갖는 형태로 전환시킬 수 있다. 이러한 당사슬 수식 효소로는 알파1,2-만노시다제, 만노시다제 A, 만노시다제 ⅠB, 만노시다제 ⅠC, 만노시다제Ⅱ, N-아세틸글루코자미닐트랜스퍼라제 Ⅰ, N-아세틸글루코자미닐트랜스퍼라제 Ⅱ, 갈락토실트랜스퍼라제, 사이알릴트랜스퍼라제, 퓨코실트랜스퍼라제 등이 포함되나, 반드시 이들로만 한정되는 것은 아니고, 만노스 잔기의 감소 및 변형에 일조할 수 있는 다양한 유전자를 사용할 수 있다. Most of the N -sugar chains attached to the glycoproteins expressed by yeast with weakened yeast-specific sugar chain biosynthetic pathways with weakened activity of the above-described Mnn1, Och1, Mnn4 and Mnn14 proteins can have a Man 8 GlcNAc 2 structure. have. When transforming the mutant strain with an expression vector capable of expressing one or more proteins having a sugar chain modification enzyme activity, it can be more effectively converted into a form having a structure similar to that of human sugar chains. These sugar chain modifying enzymes include
따라서, 또 다른 양태로서, 본 발명은 당사슬 수식 효소의 발현 벡터를 추가로 포함하는 효모 변이주를 제공한다. 바람직하게는 당사슬 수식 효소는 알파 1, 2-만노시다제, 만노시다제 ⅠA, 만노시다제 ⅠB, 만노시다제 ⅠC, 만노시다제Ⅱ, N-아세틸글루코자미닐트랜스퍼라제 Ⅰ, N-아세틸글루코자미닐트랜스퍼라제 Ⅱ, 갈락토실트랜스퍼라제, 사이알릴트랜스퍼라제, 퓨코실트랜스퍼라제 등로 이루어진 그룹 중에서 선택될 수 있다.Accordingly, as another aspect, the present invention provides a yeast mutant strain further comprising an expression vector of a sugar chain modification enzyme. Preferably, the sugar chain modification enzyme is
또한, 상기 재조합 효모 균주는 당단백질을 코딩하는 유전자를 추가로 포함할 수 있다. In addition, the recombinant yeast strain may further include a gene encoding a glycoprotein.
이를 위하여 상기 당단백질을 코딩하는 유전자를 포함하는 재조합 벡터가 상기 효모 균주에 도입된 것일 수 있다. To this end, a recombinant vector including a gene encoding the glycoprotein may be introduced into the yeast strain.
상기 “재조합 벡터”는 적합한 숙주 내에서 목적 단백질을 발현시킬 수 있도록 적합한 조절 서열에 작동 가능하게 연결된 상기 목적 단백질을 암호화하는 폴리뉴클레오티드의 염기서열을 함유하는 DNA 생산물을 의미하며, 특히 작동 가능하도록 연결된 목적 단백질을 코딩하는 유전자의 발현을 지시할 수 있는데, 이러한 벡터를 발현 벡터라고 한다.The “recombinant vector” refers to a DNA product containing a nucleotide sequence of a polynucleotide encoding the protein of interest operably linked to a suitable regulatory sequence so that the protein of interest can be expressed in a suitable host, and in particular, operably linked Expression of a gene encoding a protein of interest can be directed, and such a vector is called an expression vector.
상기 발현 벡터에는, 목적 유전자의 발현의 억제 또는 증폭, 또는 유도를 위한 각종의 기능을 가진 발현 억제용의 단편이나, 형질전환체의 선택을 위한 마커나 항생물질에 대한 내성 유전자, 균체 밖으로의 분비를 목적으로 한 시그널을 코딩하는 유전자, 난발현성 단백질에 적합한 맞춤형 융합인자 등을 추가로 포함할 수 있다.In the expression vector, a fragment for suppressing expression having various functions for suppressing or amplifying or inducing the expression of a target gene, a marker for selection of a transformant, a resistance gene for antibiotics, and secretion outside the cells It may further include a gene encoding a signal for the purpose of, a customized fusion factor suitable for a poorly expressed protein, and the like.
목적하는 당단백질을 코딩하는 유전자를 포함하는 본 발명에 따른 상기 효모 균주를, 상기 목적하는 당단백질을 발현하는데 적합한 배양 조건 및 배지에서 배양하여 당단백질을 생산하는 경우, 천연형 효모 균주를 이용하여 목적하는 당단백질을 생산하는 경우에 비하여 만노스인산의 부가가 감소 또는 완전히 제어되어 인체에 대한 면역반응을 유발할 가능성이 낮은 재조합 당단백질을 제조할 수 있는 이점을 가진다. When producing a glycoprotein by culturing the yeast strain according to the present invention containing a gene encoding a desired glycoprotein in a culture condition and medium suitable for expressing the desired glycoprotein, using a natural yeast strain Compared to the case of producing the desired glycoprotein, the addition of mannose phosphate is reduced or completely controlled, and thus it has the advantage of being able to produce a recombinant glycoprotein that is less likely to induce an immune response to the human body.
상기 목적하는 당단백질은 상기 효모 균주에서 발현시키고자 하는 당단백질이라면 특별히 그 종류는 제한되지 않으며, 병원체 단백질(pathogen protein), 성장 인자(growth factor), 사이토카인(cytokine, 예: 인터페론-α, 인터페론-β, 인터페론-γ, G-CSF 등), 케모카인(chemokine), 응집인자(예: VⅢ 인자, Ⅸ 인자, 인간 단백질 C), 내피성장인자, 성장호르몬 방출인자, HIV 외피 단백질(HIV envelope protein), 인플루엔자 바이러스 A 헤마글루티닌(influenza virus A haemagglutinin), 인플루엔자 뉴라미니다제(influenza neuraminidase), 소의 엔테로카이네이즈(enterokinase) 활성인자, 소의 포진 바이러스 타입-1 당단백질 D(Bovine herpes virus type-1 glycoprotein D), 인간 안지오스타틴(human angiostatin), 인간 B7-1, B7-2및 B-7 수용체 CTLA-4, 인간 조직 인자(human tissue factor), 성장 인자(예: 혈소판-유래 성장 인자), 인간 α-앤티트립신(human α-antitrypsin), 인간 에리트로포이에틴, 조직플라즈미노겐 활성화인자(tissue plasminogen activator), 플라즈미노겐 활성화인자 억제인자-1(plasminogen activator inhibitor-1), 우로키나제(urokinase), 플라즈미노겐, 트롬빈, 항체 또는 이의 항원-결합 단편(antigen binding fragment), 또는 융합 단백질(fusion protein) 등 제조하고자 하는 단백질이라면 어떠한 종류라도 사용할 수 있다. 또한, 상기 목적하는 당단백질의 예로서, Gas1 (beta-1,3-glucanosyltransferase), 글루코세레브로시다제(Glucocerebrosidase, GCase), 알파-갈락토시다제(alpha-galactosidase), 알파-글루코시데이즈(alpha-glucosidase), 아이두로니데이즈(iduronidase), 아이두로네이트 설페테이즈(Iduronidase sulfatase) 및 GalNAc 설페테이즈(sulfatase) 등을 들 수 있으나, 이에 제한되지 않는다. The type of the glycoprotein of interest is not particularly limited as long as it is a glycoprotein to be expressed in the yeast strain, and pathogen protein, growth factor, cytokine (e.g., interferon-α, Interferon-β, interferon-γ, G-CSF, etc.), chemokine, aggregation factor (e.g., factor VIII, factor IX, human protein C), endothelial growth factor, growth hormone release factor, HIV envelope protein), influenza virus A haemagglutinin, influenza neuraminidase, bovine enterokinase activator, bovine herpes virus type-1 glycoprotein D (Bovine herpes virus type) -1 glycoprotein D), human angiostatin, human B7-1, B7-2 and B-7 receptor CTLA-4, human tissue factor, growth factor (e.g. platelet-derived growth factor) , Human α-antitrypsin, human erythropoietin, tissue plasminogen activator, plasminogen activator inhibitor-1, urokinase ( urokinase), plasminogen, thrombin, antibodies or antigen-binding fragments thereof, or fusion proteins. In addition, as examples of the glycoprotein of interest, Gas1 (beta-1,3-glucanosyltransferase), glucocerebrosidase (GCase), alpha-galactosidase (alpha-galactosidase), alpha-glucosidase (alpha-glucosidase), iduronidase, iduronate sulfatase, GalNAc sulfatase, and the like, but are not limited thereto.
또 하나의 양태로서, 본 발명은 상기 재조합 효모 균주를 이용하여 재조합 당단백질을 제조하는 방법을 제공한다. As another aspect, the present invention provides a method for producing a recombinant glycoprotein using the recombinant yeast strain.
구체적으로, 본 발명은 Specifically, the present invention
(a) 재조합 당단백질을 코딩하는 유전자를 포함하는, Mnn4 단백질 및 서열번호 1로 표시되는 Mnn14 단백질의 활성이 모두 내재적 활성에 비하여 약화된, 재조합 효모 균주를 배양하여 상기 당단백질을 생산하는 단계; 및 (a) producing the glycoprotein by culturing a recombinant yeast strain, in which the activities of both the Mnn4 protein and the Mnn14 protein represented by SEQ ID NO: 1, including the gene encoding the recombinant glycoprotein, are weakened compared to the intrinsic activity; And
(b) 상기 (a) 단계에서 생산된 당단백질을 회수하는 단계를 포함하는, 재조합 당단백질을 제조하는 방법을 제공한다. (b) it provides a method for producing a recombinant glycoprotein comprising the step of recovering the glycoprotein produced in step (a).
상기 효모 균주, 재조합 당단백질, Mnn4 단백질, Mnn14 단백질, 내재적 활성에 비하여 약화 등에 대해서는 앞서 설명한 바와 같다. The yeast strain, recombinant glycoprotein, Mnn4 protein, Mnn14 protein, weakening compared to intrinsic activity, etc. are as described above.
상기 (a) 단계의 배양은 목적하는 당단백질을 생산할 수 있는 배양 조건 및 배지 조건임이 바람직하며, 이는 당업자가 적절히 조정할 수 있다. The culture in step (a) is preferably a culture condition and a medium condition capable of producing a desired glycoprotein, which can be appropriately adjusted by a person skilled in the art.
상기 (b) 단계는 생산된 당단백질을 배양된 세포 또는 이의 상등액에서 회수하는 단계일 수 있으며, 크로마토그래피 등의 공정을 포함할 수 있으나, 이에 제한되지 않으며, 회수에 적절한 과정을 당업자가 선택할 수 있다.The step (b) may be a step of recovering the produced glycoprotein from cultured cells or a supernatant thereof, and may include a process such as chromatography, but is not limited thereto, and a person skilled in the art can select a process suitable for recovery. have.
또한, 상기 방법은 회수된 단백질을 분리 정제하는 과정 등이 추가로 포함될 수 있다. In addition, the method may further include a process of separating and purifying the recovered protein.
또 하나의 양태로서, 본 발명은 만노스 인산이 부가되지 않은 재조합 당단백질의 제조에 사용하기 위한 상기 재조합 효모 균주의 용도를 제공한다. In another aspect, the present invention provides the use of the recombinant yeast strain for use in the production of a recombinant glycoprotein to which mannose phosphate is not added.
상기 당단백질, 효모 균주 등에 대해서는 앞서 설명한 바와 같다. The glycoprotein, yeast strain, and the like are as described above.
또 하나의 양태로서, 본 발명은 당단백질에서 만노스 인산의 부가를 제어하는 것에 사용하기 위한, 서열번호 1로 표시되는 단백질의 용도를 제공한다.In another aspect, the present invention provides the use of a protein represented by SEQ ID NO: 1 for use in controlling the addition of mannose phosphate in a glycoprotein.
상기 서열번호 1로 표시되는 단백질에 대해서는 앞서 설명한 바와 같다. The protein represented by SEQ ID NO: 1 is as described above.
또 하나의 양태로서, 본 발명은 만노스인산화에 사용하기 위한, 서열번호 1로 표시되는 단백질의 용도를 제공한다.In another aspect, the present invention provides the use of the protein represented by SEQ ID NO: 1 for use in mannose phosphorylation.
상기 서열번호 1로 표시되는 단백질에 대해서는 앞서 설명한 바와 같다. The protein represented by SEQ ID NO: 1 is as described above.
재조합 당단백질의 제조 시 본 발명에 따른 재조합 효모 균주를 이용하면 효모 특이적인 만노스인산의 부가를 제거 함으로서 인체 주입 시 면역 반응을 유발하지 않는 보다 인간화된 당사슬이 부착된 당단백질을 제조할 수 있다. When the recombinant yeast strain according to the present invention is used in the manufacture of a recombinant glycoprotein, a more humanized sugar chain attached glycoprotein can be prepared that does not induce an immune response during human injection by removing the addition of yeast-specific mannose phosphate.
도 1은 och1Δmnn1Δ 균주로부터 MNN4 유전자를 결손 하는 실험 결과이다. (A) Mnn4_F와 Mnn4_R 프라이머를 이용하여 pUG73으로부터 loxP - LEU2 - loxP 카세트를 중합효소 연쇄반응으로 증폭하여 2.5 kbp의 DNA 절편을 얻었다. (B) 준비된 결손 카세트를 och1Δmnn1Δ 균주에 도입하고 SC-Leu 배지에서 선별한 형질전환체들의 게노믹 DNA로부터 확인용 프라이머인 Leu2-CF와 Mnn4-CR들을 이용하여 MNN4 유전자가 LEU2 유전자로 교체된 1.8 kbp의 DNA 절편을 증폭하여 MNN4의 결손을 확인한 결과이다.
도 2는 och1Δmnn1Δmnn4Δ 균주로부터 MNN6 유전자를 결손 하는 실험 결과이다. (A) Mnn6_F와 Mnn6_R 프라이머를 이용하여 pUG72로부터 loxP - URA3 - loxP 카세트를 중합효소 연쇄반응으로 증폭하여 1.7 kbp의 DNA 절편을 얻었다. (B) 준비된 결손 카세트를 och1Δmnn1Δmnn4Δ 균주에 도입하고 SC-Ura 배지에서 선별한 형질전환체들의 게노믹 DNA로부터 확인용 프라이머인 Mnn6-CF와 Ura3-CR을 이용하여 MNN6 유전자가 URA3 유전자로 교체된 2.4 kbp의 DNA 절편을 증폭하여 MNN6의 결손을 확인한 결과이다.
도 3은 och1Δmnn1Δ 균주와 MNN4와 MNN6 유전자가 추가 결손 된 균주들의 CWMs로부터 얻어진 N-당사슬을 DNA 시퀀서를 이용한 분석한 결과이다. 상대적인 위치 확인을 위해서 포도당 유닛(glucose unit)을 나타내는 말토덱스트린 레퍼런스(maltodextrin reference) 프로파일을 맨 처음 패널에 보여주었다(Dex). 모든 결손 균주(och1Δmnn1Δ, och1Δmnn1Δmnn4Δ, och1Δmnn1Δmnn4Δmnn6Δ)들의 프로파일에서 (Man-P)2-Man8GlcNAc2, Man-P-Man8GlcNAc2 및 Man8GlcNAc2의 세 개의 당사슬 피크들이 주요하게 관찰되었다. 이들은 미국 Consortium for Functional Glycomics (htpp://www.functionalglycomics.org/)의 기호를 이용하여 표시하였다(청색 네모: GlcNAc, 녹색 원: 만노스, 인산: P). 그리고 동정이 되지 않은 피크들은 *로 표시하였다.
도 4는 Mnn6 단백질과 이들 단백질 사이의 동일성과 상동성 분석 결과를 나타낸 것이다.
도 5는 och1Δmnn1Δmnn4Δmnn6Δ 균주로부터 추가로 MNN4와 MNN6의 상동 유전자들을 결손 하는 실험 결과이다. (A) MNN14 , YUR1 , KTR2 , KTR4 , KTR5와 KTR7 유전자 결손 카세트 제작을 위해서 해당 유전자의 5`과 3`-UTR과 상동성 부위를 가진 프라이머들들을 이용하여 pUG72 벡터로부터 loxP - URA3 - loxP 카세트를 중합효소 연쇄반응으로 증폭하여 1.7 kbp의 DNA 절편을 얻었다. (B) 준비된 결손 카세트를 och1Δmnn1Δmnn4Δmnn6Δ 균주에 도입하고 SC-Ura 배지에서 선별한 형질전환체들의 게노믹 DNA로부터 확인용 프라이들을 이용하여 해당 유전자가 URA3 유전자로 교체된 1.2 kbp의 DNA 절편을 증폭하여 각각의 결손을 확인한 결과이다.
도 6은 최소 배지에서 배양한 och1Δmnn1Δmnn4Δmnn6Δ 균주 및 MNN4 또는 MNN6의 상동 유전자들이 추가로 결손된 균주들의 CWMs로부터 얻은 N-당사슬의 분석 결과이다. 도 3에서와 마찬가지로 상대적인 위치 확인을 위한 말토덱스트린 레퍼런스를 맨 처음 패널에 두었다(Dex). 그 다음 패널부터 차례로 och1Δmnn1Δmnn4Δ mnn6Δ, och1Δmnn1Δmnn4Δmnn6Δyur1Δ (- yur1Δ), och1Δmnn1Δmnn4Δmnn6Δ ktr2Δ (- ktr2Δ), och1Δmnn1Δmnn4Δmnn6Δktr4Δ (- ktr4Δ), och1Δmnn1Δmnn4 Δmnn6Δktr5Δ (- ktr5Δ), och1Δmnn1Δmnn4Δmnn6Δktr7Δ (- ktr7Δ) 및 och1Δ mnn1Δmnn4Δmnn6Δmnn14Δ (- mnn14Δ) 균주들의 N-당사슬 프로파일을 보여준다. 당사슬 피크들은 도 3과 동일한 방식으로 표시하였다.
도 7은 YPD 배지에서 배양한 och1Δmnn1Δmnn4Δmnn6Δ 균주 및 MNN4 또는 MNN6의 상동 유전자들이 추가로 결손 된 균주들의 CWMs로부터 얻은 N-당사슬의 분석 결과이다. 도 6과 동일한 방법으로 패널의 순서와 당사슬 피크들을 나타내었다.
도 8은 MNN14 유전자가 결손 된 균주들의 CWMs로부터 얻은 N-당사슬의 분석 결과이다. 도 3에서와 마찬가지로 상대적인 위치 확인을 위한 말토덱스트린 레퍼런스를 맨 처음 패널에 두었다(Dex). 그 다음 패널부터 차례로 och1Δmnn1Δmnn4Δ mnn6Δmnn14Δ, och1Δmnn1Δmnn4Δmnn14Δ , och1Δmnn1Δmnn14Δ 균주들의 N-당사슬 프로파일을 보여준다. 당사슬 피크들은 도 3과 동일한 방식으로 표시하였다.
도 9는 만노스인산화 능력이 제거되었던 och1Δmnn1Δmnn4Δmnn14Δ 균주에 MNN4 또는 MNN14 유전자를 발현하여 만노스인산화 능력의 회복 여부를 보는 complementation 실험 결과이다. 도 3에서와 마찬가지로 상대적인 위치 확인을 위한 말토덱스트린 레퍼런스를 맨 처음 패널에 두었다(Dex). 그 다음 패널부터 차례로 YEp352-GAP (Mock), YEp352-Mnn4 (Mnn4) 또는 YEp352-Mnn14 (Mnn14) 벡터로 형질전환한 균주들의 N-당사슬 프로파일을 보여준다. 당사슬 피크들은 도 3과 동일한 방식으로 표시하였다.
도 10은 만노스인산화 능력이 제거되었던 och1Δmnn1Δmnn4Δmnn6Δmnn14Δ 균주에 MNN4 또는 MNN14 유전자를 발현하여 만노스인산화 능력의 회복 여부를 보는 complementation 실험 결과이다. 도 9와 동일한 방법으로 패널의 순서와 당사슬 피크들을 나타내었다.
도 11은 och1Δmnn1Δmnn4Δmnn6Δ , och1Δmnn1Δmnn4Δmnn14Δ , och1Δmnn1 Δmnn4Δmnn14Δ/Mnn4 및 och1Δmnn1Δmnn4Δmnn14Δ / Mnn14 균주에서 분비 발현한 재조합 Gas1 단백질을 정제하여 그 N-당사슬 프로파일을 분석한 실험 결과이다. 각 당사슬 피크들은 도 3과 동일한 방식으로 표시하였다.
도 12는 och1Δmnn1Δmnn4Δmnn6Δ , och1Δmnn1Δmnn4Δmnn14Δ , och1Δmnn1 Δmnn4Δmnn14Δ/Mnn4 및 och1Δmnn1Δmnn4Δmnn14Δ / Mnn14 균주에서 분비 발현한 재조합 Gas1 단백질을 정제하여 그 등전점을 isoelectric focusing (IEF) 방법을 이용하여 분석한 결과를 보여준다.1 is an experimental result of deletion of the MNN4 gene from the och1Δmnn1Δ strain. (A) A 2.5 kbp DNA fragment was obtained by amplifying the loxP - LEU2 - loxP cassette from pUG73 by polymerase chain reaction using Mnn4_F and Mnn4_R primers. (B) 1.8 in which the prepared deletion cassette was introduced into the och1Δmnn1Δ strain, and the MNN4 gene was replaced with the LEU2 gene using the identification primers Leu2-CF and Mnn4-CRs from the genomic DNA of the transformants selected in SC-Leu medium. This is the result of confirming the deletion of MNN4 by amplifying the kbp DNA fragment.
2 is an experimental result of deletion of the MNN6 gene from the och1Δmnn1Δmnn4Δ strain. (A) A DNA fragment of 1.7 kbp was obtained by amplifying the loxP - URA3 - loxP cassette from pUG72 by polymerase chain reaction using Mnn6_F and Mnn6_R primers. (B) The prepared deletion cassette was introduced into the och1Δmnn1Δmnn4Δ strain, and the MNN6 gene was replaced with the URA3 gene using the identification primers Mnn6-CF and Ura3-CR from the genomic DNA of the transformants selected in SC-Ura medium 2.4. This is the result of confirming the deletion of MNN6 by amplifying the kbp DNA fragment.
3 is an analysis result of the N -sugar chain obtained from the och1Δmnn1Δ strain and the CWMs of strains in which the MNN4 and MNN6 genes are additionally deleted, using a DNA sequencer. To determine the relative position, a maltodextrin reference profile representing glucose units was shown in the first panel (Dex). In the profile of all the defective strains ( och1Δmnn1Δ , och1Δmnn1Δmnn4Δ , och1Δmnn1Δmnn4Δmnn6Δ ), three sugar chain peaks of (Man-P) 2 -Man 8 GlcNAc 2 , Man-P-Man 8 GlcNAc 2 and Man 8 GlcNAc 2 were observed mainly. These were indicated using the symbols of the US Consortium for Functional Glycomics (htpp://www.functionalglycomics.org/) (blue square: GlcNAc, green circle: mannose, phosphoric acid: P). And peaks that were not identified are marked with *.
4 shows the results of analysis of identity and homology between the Mnn6 protein and these proteins.
5 is an experimental result of additionally deleting homologous genes of MNN4 and MNN6 from the och1Δmnn1Δmnn4Δmnn6Δ strain. (A) MNN14, YUR1, KTR2 , KTR4, KTR5 and KTR7 gene defect cassette for production using the 5` and 3`-UTR and a primer having a homologous region of the gene from a vector pUG72 loxP - URA3 - loxP cassette Was amplified by polymerase chain reaction to obtain a 1.7 kbp DNA fragment. (B) The prepared deletion cassette was introduced into the och1Δmnn1Δmnn4Δmnn6Δ strain, and a 1.2 kbp DNA fragment in which the corresponding gene was replaced with the URA3 gene was amplified from the genomic DNA of the transformants selected in SC-Ura medium. This is the result of confirming the deficit of.
6 is an analysis result of the N -sugar chain obtained from CWMs of och1Δmnn1Δmnn4Δmnn6Δ strains cultured in a minimal medium and strains in which homologous genes of MNN4 or MNN6 are additionally deleted. As in FIG. 3, a maltodextrin reference for checking the relative position was first placed on the panel (Dex). From the next panel in turn och1Δmnn1Δmnn4Δ mnn6Δ, och1Δmnn1Δmnn4Δmnn6Δyur1Δ (- yur1Δ), och1Δmnn1Δmnn4Δmnn6Δ ktr2Δ ( - ktr2Δ), och1Δmnn1Δmnn4Δmnn6Δktr4Δ (- ktr4Δ), och1Δmnn1Δmnn4 Δmnn6Δktr5Δ ( - ktr5Δ), och1Δmnn1Δmnn4Δmnn6Δktr7Δ (- ktr7Δ) and och1Δ mnn1Δmnn4Δmnn6Δmnn14Δ (- mnn14Δ) - shows the oligosaccharide profile of the strain N. Oligosaccharide peaks were expressed in the same manner as in FIG. 3.
7 is an analysis result of the N -sugar chain obtained from CWMs of och1Δmnn1Δmnn4Δmnn6Δ strains cultured in YPD medium and strains in which homologous genes of MNN4 or MNN6 are additionally deleted. The order of the panel and sugar chain peaks were shown in the same manner as in FIG. 6.
8 is an analysis result of the N -sugar chain obtained from CWMs of strains in which the MNN14 gene is deleted. As in FIG. 3, a maltodextrin reference for checking the relative position was first placed on the panel (Dex). The following panel shows the N -sugar chain profiles of och1Δmnn1Δmnn4Δ mnn6Δmnn14Δ, och1Δmnn1Δmnn4Δmnn14Δ and och1Δmnn1Δmnn14Δ strains in order from the next panel. Oligosaccharide peaks were expressed in the same manner as in FIG. 3.
9 is a result of a complementation experiment to see whether the mannose phosphorylation ability is restored by expressing the MNN4 or MNN14 gene in the och1Δmnn1Δmnn4Δmnn14Δ strain from which the mannose phosphorylation ability has been removed. As in FIG. 3, a maltodextrin reference for checking the relative position was first placed on the panel (Dex). From the next panel, the N -sugar chain profiles of strains transformed with YEp352-GAP (Mock), YEp352-Mnn4 (Mnn4) or YEp352-Mnn14 (Mnn14) vector in turn are shown. Oligosaccharide peaks were expressed in the same manner as in FIG. 3.
10 is a result of a complementation experiment to see whether the mannose phosphorylation ability is recovered by expressing the MNN4 or MNN14 gene in the och1Δmnn1Δmnn4Δmnn6Δmnn14Δ strain from which the mannose phosphorylation ability has been removed. The order of the panel and sugar chain peaks were shown in the same manner as in FIG. 9.
Figure 11 Purification of the secreted expression of a Gas1 protein in recombinant och1Δmnn1Δmnn4Δmnn6Δ, och1Δmnn1Δmnn4Δmnn14Δ, och1Δmnn1 Δmnn4Δmnn14Δ / Mnn4 and och1Δmnn1Δmnn4Δmnn14Δ / Mnn14 strains that N - an experimental analysis of the sugar chain profile. Each oligosaccharide peak was expressed in the same manner as in FIG. 3.
Figure 12 shows the result of isoelectric point analysis by purifying the recombinant Gas1 protein secreted and expressed in och1Δmnn1Δmnn4Δmnn6Δ, och1Δmnn1Δmnn4Δmnn14Δ , och1Δmnn1 Δmnn4Δmnn14Δ/Mnn4 and och1Δmnn1Δmnn4Δmnn14Δ / Mnn14 strains.
이하 본 발명을 하기 예에 의해 상세히 설명한다. 다만, 하기 예는 본 발명을 예시하기 위한 것일 뿐, 하기 예에 의하여 본 발명의 범위가 제한되는 것은 아니다. Hereinafter, the present invention is described in detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited by the following examples.
실시예Example 1. One. S. S. cerevisiaecerevisiae och1Δmnn1Δoch1Δmnn1Δ 균주에서 From strain MNN4MNN4 및 And MNN6MNN6 유전자의 결손 Gene defect
1-1: S. cerevisiae och1Δmnn1Δ 균주의 제조1-1: S. cerevisiae Preparation of och1Δmnn1Δ strain
S. cerevisiae och1Δmnn1Δ 균주는 L3262 균주로부터 Cre/loxP 시스템을 이용한 다중 유전자 결손 방법(U. Gueldener et al, 2002, Nucleic Acids Res 30: e23; J.H. Hegemann, S.B. Heick, 2011, Methods Mol Biol 765: 189-206)을 이용하여 제작하였다. 우선, ScOCH1 유전자를 결손하기 위한 loxP - URA3 - loxP 결손 카세트를 Och1_pUG72_F와 Och1_pUG72_R 프라이머를 이용하여 pUG72 벡터로부터 증폭하여 제작하였다. 1 M 소르비톨(sorbitol)이 첨가된 SC-Ura 배지에서 형질전환된 균주들을 선별한 후, Och1_CF과 Och1_CR 프라이머들을 이용한 염색체 DNA를 주형으로 한 중합효소 연쇄반응을 이용하여 ScOCH1 유전자 결손을 확인하였다. 이어서 ScMNN1 유전자 결손을 위한 loxP - kanMX - loxP 카세트를 pUG6를 주형으로 하고, Mnn1_pUG6_F와 Mnn1_pUG6_R 프라이머들을 이용하여 제작하였으며, 상기 제작된 och1Δ 균주에 형질전환하였다. 1 M 소르비톨(sorbitol)과 200 mg/L G418이 첨가된 YPD 배지에서 형질전환된 균주를 선별하였으며, ScMNN1 유전자 결손의 확인은 염색체 DNA를 주형으로 한, Mnn1_CF와 Mnn1_CR 프라이머들을 이용한 중합효소 연쇄반응을 이용하여 확인하였다. 그리고, 유전자들의 결손을 위해서 들어간 선별용 마커들은 상기의 논문들에 보고된 바와 같이, pSH68 벡터를 형질전환하고 2% 포도당 대신에 2% 갈락토스가 첨가된 SC-Leu 배지에서 Cre 단백질을 발현시켜서 제거하였다. 마지막으로 Cre 단백질 발현을 위한 pSH68는 YPD 배지에서 연속 배양을 통해서 제거하였으며, 적절한 선별 배지에서의 성장을 확인하여 최종 mnn1Δoch1Δ 균주를 선별하였다. S. cerevisiae The och1Δmnn1Δ strain is a multiple gene deletion method using the Cre/loxP system from the L3262 strain (U. Gueldener et al , 2002, Nucleic Acids Res 30: e23; JH Hegemann, SB Heick, 2011, Methods Mol. Biol 765: 189-206). First, a loxP - URA3 - loxP deletion cassette for deletion of the ScOCH1 gene was amplified from the pUG72 vector using Och1_pUG72_F and Och1_pUG72_R primers. After selecting the transformed strains in SC-Ura medium to which 1 M sorbitol was added, the ScOCH1 gene deletion was confirmed using a polymerase chain reaction using chromosomal DNA as a template using Och1_CF and Och1_CR primers. Subsequently , a loxP - kanMX - loxP cassette for ScMNN1 gene deletion was prepared using pUG6 as a template, and Mnn1_pUG6_F and Mnn1_pUG6_R primers were used, and the och1Δ strain was transformed. Transformed strains were selected in YPD medium supplemented with 1 M sorbitol and 200 mg/L G418, and the ScMNN1 gene deletion was confirmed by polymerase chain reaction using Mnn1_CF and Mnn1_CR primers using chromosomal DNA as a template. It was confirmed using. And, as reported in the above papers, the selection markers entered for deletion of genes were removed by transforming the pSH68 vector and expressing the Cre protein in SC-Leu medium to which 2% galactose was added instead of 2% glucose. I did. Finally, pSH68 for Cre protein expression was removed through continuous culture in YPD medium, and growth was confirmed in an appropriate selection medium to select the final mnn1Δoch1Δ strain.
상기 명명된 프라이머의 서열들은 하기와 같았다. The sequences of the named primers were as follows.
1-2: S. cerevisiae och1Δmnn1Δ 균주에서 MNN4 또는 MNN6 유전자의 결손1-2: S. cerevisiae Deletion of MNN4 or MNN6 gene in och1Δmnn1Δ strain
상기 실시예 1-1.에서 제작하였던 S. cerevisiae L3262 och1Δmnn1Δ 균주로부터 Cre/loxP 시스템을 이용한 다중 유전자 결손 방법(U. Gueldener et al, 2002, Nucleic Acids Res 30: e23; J.H. Hegemann, S.B. Heick, 2011, Methods Mol Biol 765: 189-206)을 이용하여 만노스인산화를 제어한다고 알려진 MNN4 유전자와 만노스인산화 효소로 알려진 MNN6 유전자를 추가로 파쇄하여 4중 결손 균주(och1Δmnn1Δmnn4Δmnn6Δ)를 제작하였다. Multiple gene deletion method using the Cre/ loxP system from the S. cerevisiae L3262 och1Δmnn1Δ strain prepared in Example 1-1 (U. Gueldener et al, 2002, Nucleic Acids Res 30: e23; JH Hegemann, SB Heick, 2011 , Methods Mol Biol 765: 189-206) was used to further disrupt the MNN4 gene known to control mannose phosphorylation and the MNN6 gene known as mannose phosphorylation enzyme to prepare a quadruple deletion strain ( och1Δmnn1Δmnn4Δmnn6Δ ).
효모 균주의 배양을 위해서는 1 M 소르비톨이 첨가된 YPD 배지(1% 효모 추출물(yeast extract), 2% 펩톤(peptone), 2% 포도당) 또는 SC(synthetic complete) 배지(0.67% 효모 질소 염기(yeast nitrogen base), 2% 포도당, 드롭아웃 아미노산 혼합물(dropout amino acid mixture), 필요한 모든 아미노산 포함) 등을 사용하여 주로 28 ℃에서 배양하였다. YPD medium (1% yeast extract, 2% peptone, 2% glucose) or SC (synthetic complete) medium (0.67% yeast nitrogen base) added with 1 M sorbitol for cultivation of yeast strains nitrogen base), 2% glucose, dropout amino acid mixture, including all necessary amino acids), etc., and incubated mainly at 28°C.
먼저, MNN4 유전자 결손을 위한 loxP - LEU2 - loxP 결손 카세트를 Mnn4_F와 Mnn4_R 프라이머(표 2)를 이용하여 pUG73 벡터로부터 중합효소 연쇄반응으로 증폭하여 2.5 kbp의 DNA 절편을 얻었다(도 1 (A)). 이렇게 얻어진 MNN4 유전자 결손 카세트를 och1Δmnn1Δ에 형질전환으로 도입하였으며, 1 M sorbitol이 첨가된 SC-LEU 선택배지(1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base w/o amino acid, DO supplement -LEU)에서 형질 전환체를 선별하였다. 그리고 선별된 형질전환체들의 염색체 DNA를 추출하고 이를 주형으로 하여 Leu2_CF와 Mnn4_CR 프라이머들을 이용한 중합효소 연쇄 반응을 통하여 MNN4 유전자의 결손을 확인하여(도 1 (B)), LEU2 선택 마커가 들어 있는 och1Δmnn1Δmnn4Δ 균주(mnn1Δ :: loxP och1Δ :: loxP mnn4Δ::loxP-LEU2-loxP)를 제작하였다.First, the loxP - LEU2 - loxP deletion cassette for MNN4 gene deletion was amplified from the pUG73 vector by polymerase chain reaction using Mnn4_F and Mnn4_R primers (Table 2) to obtain a DNA fragment of 2.5 kbp (Fig. 1 (A)). . The thus obtained MNN4 gene deletion cassette was transformed into och1Δmnn1Δ , and 1 M sorbitol was added to the SC-LEU selective medium (1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base w/o amino acid, DO supplement -LEU). ) Was selected for transformants. Then, the chromosomal DNA of the selected transformants was extracted and used as a template to confirm the deletion of the MNN4 gene through polymerase chain reaction using Leu2_CF and Mnn4_CR primers (Fig. 1(B)), and och1Δmnn1Δmnn4Δ containing the LEU2 selection marker. Strain ( mnn1Δ :: loxP och1Δ :: loxP mnn4Δ::loxP-LEU2-loxP ) was prepared.
MNN6 유전자 결손을 위한 loxP - URA3 - loxP 결손 카세트는 Mnn6_F와 Mnn6_R 프라이머(표 2)를 이용하여 pUG72 벡터로부터 중합효소 연쇄반응으로 증폭하여 1.7 kbp의 DNA 절편을 얻었다(도2 (A)). 이렇게 얻어진 MNN6 유전자 결손 카세트를 상기에서 제작한 och1Δmnn1Δmnn4Δ 균주에 형질전환으로 도입하였으며, 1 M 소르비톨이 첨가된 SC-LEU 선택배지에서 형질 전환체를 선별하였다. 그리고 선별된 형질 전환체들의 염색체 DNA를 추출하고 이를 주형으로 하여 Mnn6_CF와 Ura3_CR 프라이머들을 이용한 중합효소 연쇄 반응을 통하여 MNN6 유전자의 결손을 확인하여(도2 (B)), LEU2와 URA3 선택마커가 들어 있는 4중 결손 och1Δmnn1Δmnn4Δmnn6Δ 균주(mnn1Δ::loxP och1Δ :: loxP mnn4Δ :: loxP - LEU2 - loxP mnn6Δ :: loxP - URA3 - loxP)를 제작하였다. The loxP - URA3 - loxP deletion cassette for the MNN6 gene deletion was amplified from the pUG72 vector by polymerase chain reaction using Mnn6_F and Mnn6_R primers (Table 2) to obtain a DNA fragment of 1.7 kbp (Fig. 2 (A)). The thus obtained MNN6 gene deletion cassette was transformed into the och1Δmnn1Δmnn4Δ strain prepared above, and transformants were selected in SC-LEU selective medium to which 1 M sorbitol was added. Then, the chromosomal DNA of the selected transformants was extracted and used as a template to confirm the deletion of the MNN6 gene through polymerase chain reaction using Mnn6_CF and Ura3_CR primers (Fig. 2(B)), and LEU2 and URA3 selection markers were included. Quadruple deletion och1Δmnn1Δmnn4Δmnn6Δ strain ( mnn1Δ::loxP och1Δ :: loxP mnn4Δ :: loxP - LEU2 - loxP mnn6Δ :: loxP - URA3 - loxP ) was prepared.
MNN4와 MNN6의 유전자 결손을 위해서 들어간 선별용 마커 LEU2와 URA3는 상기의 논문들에 보고된 바와 같이, 해당 균주들에 Cre 단백질 발현을 위한 pSH67 벡터를 도입하고 YPDS-G418 선택배지(1 M sorbitol, 2% glucose, 1% yeast extrat, 2% peptone, 200㎍/㎖ G418)에서 형질 전환체를 선별한 후, 이를 2% 포도당 대신 2% 갈락토오스(galactose)가 첨가된 YPDS-G418 배지에서 배양하여 Cre 단백질 발현시켜서 제거하였다. 그리고 Cre 단백질 발현을 위한 pSH47 벡터는 YPD 배지에서 연속 배양을 통해서 제거하였으며, 적절한 선별 배지에서의 성장을 확인하여 LEU2 마커가 제거된 3중 결손 och1Δmnn1Δmnn4Δ균주(mnn1Δ :: loxP och1Δ :: loxP mnn4Δ ::loxP)와 LEU2와 URA3선택 마커가 제거된 och1Δmnn1Δmnn4Δmnn6Δ 균주(mnn1Δ ::loxP och1Δ::loxP mnn4Δ::loxP mnn6Δ::loxP)를 제작하였다. MNN4 and the selectable marker LEU2 and URA3 for entering to the genetic defect of MNN6 are as reported in the above paper, introducing pSH67 vector for Cre protein expression in the strain, and YPDS-G418 selection medium (1 M sorbitol, After selecting transformants from 2% glucose, 1% yeast extrat, 2% peptone, 200㎍/㎖ G418), they were cultured in YPDS-G418 medium supplemented with 2% galactose instead of 2% glucose. It was removed by protein expression. And the pSH47 vector for Cre protein expression was removed through continuous culture in YPD medium, and the triple deletion och1Δmnn1Δmnn4Δ strain from which the LEU2 marker was removed by confirming growth in an appropriate selection medium (mnn1Δ :: loxP och1Δ :: loxP the mnn4Δ :: loxP) and the LEU2 and URA3 selection marker is removed och1Δmnn1Δmnn4Δmnn6Δ strain (mnn1Δ :: loxP och1Δ :: loxP mnn4Δ :: loxP mnn6Δ :: loxP) was produced.
실시예Example 2. 2. och1Δmnn1Δoch1Δmnn1Δ 균주 및 Strain and MNN4MNN4 와Wow MNN6MNN6 가end 추가 결손 된 균주들의 Of strains with additional deletions NN -- 당사슬Our chain 비교 compare
상기 제작된 och1Δmnn1Δ 균주와 3중 결손 och1Δmnn1Δmnn4Δ 균주 및 4중 결손 och1Δmnn1Δmnn4Δmnn6Δ 균주들의 N-당사슬을 비교 분석하여 만노스 인산의 부가 효율의 변화를 관찰하였다. The N -sugar chains of the prepared och1Δmnn1Δ strain, the triple-defective och1Δmnn1Δmnn4Δ strain and the quadruple-defect och1Δmnn1Δmnn4Δmnn6Δ strain were compared and analyzed to observe the change in the addition efficiency of mannose phosphate.
2-1: DNA 시퀀서를 이용한 효모 세포 벽 만노단백질(cell wall mannoproteins, CWMs)의 N-당사슬 분석 2-1: Analysis of N -sugar chains of yeast cell wall mannoproteins (CWMs) using a DNA sequencer
우선 효모의 CWMs을 기존에 보고된 핫 구연산 버퍼(hot citrate buffer: 20 mM sodium citrate buffer, pH 7.0)를 이용한 방법을 따라서 추출하고(Park et al, 2011, Appl Environ Microbiol 77: 1187-1195), DNA 시퀀서를 이용한 방법으로 분석하였다(Laroy et al, 2006, Nat Protoc 1: 397-405). First, yeast CWMs were extracted according to the method using a previously reported hot citrate buffer (20 mM sodium citrate buffer, pH 7.0) (Park et al, 2011, Appl Environ Microbiol). 77: 1187-1195), and analyzed by a method using a DNA sequencer (Laroy et al, 2006, Nat Protoc 1: 397-405).
간단히 기술하면, 2 ㎎의 CWMs에 2 배 부피의 Membrane Denaturing 버퍼 (MDB; 8 M Urea, 360 mM Tris-HCl (pH 8.6), 3.2 mM EDTA)를 첨가한 후 50 ℃에서 한 시간 방치하여 denaturation을 시켰다. 그리고 이를 메탄올로 활성화 시키고, 증류수로 씻어준 MultiScreen-immobilon PVDF membrane clear plate (Millipore, Billerica, MA, USA)에 로딩하였다. 0.1 M 디티오트레이톨(dithiothreitol)이 녹아 있는 MDB를 처리하여 37 ℃에서 1시간 반응 후 증류수로 씻어주고, 0.1 M 아이오도아세트산(iodoacetic acid)이 있는 MDB를 첨가하여 실온의 암 상태에서 30분 반응한 후, 증류수로 씻어주었다. 이후 증류수에 녹인 1% 폴리비닐피롤리돈(polyvinylpyrrolidone) 360 이용하여 멤브레인을 블록킹한 후, 증류수로 씻어주었다. 10 mM 트리스-아세테이트(tris-acetate) (pH 8.3)에 펩타이드 N-글리코시다제 F(PNGase F; New England Biolabs, MA, USA)를 처리하여 37 ℃에서 16시간 반응하여 N-당사슬을 절단 하여 회수하고, 이를 진공 건조시켰다. 그리고 100 mM APTS(8-Aminopyrene-1,3,6-trisulfonic acid) 를 1.2 M 구연산(citric acid)과 섞어 최종 20 mM APTS를 만들고 DMSO에 녹인 1 M 소듐 시아노브롬하이드리드(sodium cyanoborohydride, NaCNBH2)를 1:1 비율로 섞은 APTS 혼합 용액을 준비하였다. 그리고 이 APTS 혼합용액을 건조된 당사슬에 5 ㎕ 첨가 하여 37 ℃에서 16시간 동안 반응시켰다. 반응 후 10 ㎕의 HPLC 증류수로 반응을 정지시켰다. Sephadex G10(GE Healthcare, Milwaukee, WI, USA) 컬럼이 패킹된 Multiscreen Durapore membrane lined 96-well plate(Millipore, Billerica, MA, USA)를 이용해서 반응을 하지 않은 과량의 APTS를 제거하여 APTS가 표지된 당사슬을 분리한 후 기존에 보고된 방법을 따라서 DNA 시퀀서를 이용하여 분석하였다(Laroy et al, Nature Protocols 1: 397-405, 2006). 즉, APTS가 표지된 당사슬 용액 10 ㎕를 DNA 시퀀서 플레이트에 옮겨 담고, POP7-polyacrylamide linear polymer로 채워진 36 cm capillary array가 장착된 ABI 3130 DNA 시퀀서를 이용하여 분석하였다. DNA 시퀀서 분석 조건은 표 3에 기재된 바와 같으며, 데이터 분석은 GeneMapper 소프트웨어를 사용하였다.Briefly, 2 times the volume of Membrane Denaturing Buffer (MDB; 8 M Urea, 360 mM Tris-HCl (pH 8.6), 3.2 mM EDTA) was added to 2 mg of CWMs and left at 50° C. for 1 hour to perform denaturation. Made it. And it was activated with methanol and loaded onto a MultiScreen-immobilon PVDF membrane clear plate (Millipore, Billerica, MA, USA) washed with distilled water. Treat MDB containing 0.1 M dithiothreitol, react at 37°C for 1 hour, rinse with distilled water, add 0.1 M iodoacetic acid, and add MDB containing 0.1 M iodoacetic acid for 30 minutes in the dark at room temperature. After reaction, it was washed with distilled water. Thereafter, the membrane was blocked using 1% polyvinylpyrrolidone 360 dissolved in distilled water, and then washed with distilled water. 10 mM tris-acetate (pH 8.3) was treated with peptide N -glycosidase F (PNGase F; New England Biolabs, MA, USA) and reacted at 37° C. for 16 hours to cleave the N -sugar chain. Recovered and vacuum dried. And 100 mM APTS (8-Aminopyrene-1,3,6-trisulfonic acid) was mixed with 1.2 M citric acid to make final 20 mM APTS, and 1 M sodium cyanoborohydride (NaCNBH2) dissolved in DMSO. ) Was mixed in a 1:1 ratio to prepare an APTS mixed solution. Then, 5 µl of this APTS mixed solution was added to the dried sugar chain and reacted at 37° C. for 16 hours. After the reaction, the reaction was stopped with 10 µl of HPLC distilled water. Sephadex G10 (GE Healthcare, Milwaukee, WI, USA) column packed Multiscreen Durapore membrane lined 96-well plate (Millipore, Billerica, MA, USA) to remove excess APTS that did not react to label APTS. After separating the sugar chain, it was analyzed using a DNA sequencer according to the previously reported method (Laroy et al, Nature Protocols 1: 397-405, 2006). That is, 10 μl of APTS-labeled oligosaccharide solution was transferred to a DNA sequencer plate, and analyzed using an ABI 3130 DNA sequencer equipped with a 36 cm capillary array filled with POP7-polyacrylamide linear polymer. DNA sequencer analysis conditions are as described in Table 3, and data analysis was performed using GeneMapper software.
2-2 : N-당사슬 분석 결과의 해석2-2: Interpretation of N -sugar chain analysis results
이중 결손 och1Δmnn1Δ, 삼중 결손 och1Δmnn1Δmnn4Δ, 사중 결손 och1Δmnn1Δmnn4Δmnn6Δ 균주들의 N-당사슬을 분석하였을 때, 모두 3개의 주요 피크들이 관찰되었다(도3). 이 피크들은 왼쪽부터 두 개의 만노스인산이 부가된 (Man-P)2-Man8GlcNAc2 당사슬, 한 개의 만노스인산이 부가된 Man-P-Man8GlcNAc2당사슬 및 중성의 Man8GlcNAc2 당사슬들이다. When analyzing the N -sugar chains of the double deletion och1Δmnn1Δ , triple deletion och1Δmnn1Δmnn4Δ , and quadruple deletion och1Δmnn1Δmnn4Δmnn6Δ strains, all three major peaks were observed (Figure 3). These peaks are from left to right: 2 -Man 8 GlcNAc 2 sugar chain to which two mannose phosphates are added, Man-P-Man 8 GlcNAc 2 sugar chain to which one mannose phosphate is added, and neutral Man 8 GlcNAc 2 These are our chains.
MNN4 유전자를 결손한 och1Δmnn1Δmnn4Δ 균주와 och1Δmnn1Δ 균주의 N-당사슬 프로파일에서 만노스인산이 부가된 당사슬의 함량을 비교하였을 때 별다른 차이를 보이지 않았다. The och1Δmnn1Δmnn4Δ strain and och1Δmnn1Δ that lack the MNN4 gene There was no significant difference when comparing the content of sugar chains to which mannose phosphate was added in the N-sugar chain profile of the strain.
MNN4와 MNN6 유전자를 함께 결손한 och1Δmnn1Δmnn4Δmnn6Δ 균주는 och1Δmnn1Δ 및 och1Δmnn1Δmnn4Δ 균주에 비해서 만노스인산이 부가된 당사슬의 함량이 다소 감소하였으나 그렇게 큰 차이를 보이지는 않았다. 이러한 결과로부터 MNN4와 MNN6 유전자 외에 다른 유전자가 만노스인산의 부가를 담당한다고 생각되었다. Och1Δmnn1Δmnn4Δmnn6Δ strains deficient with the MNN4 and MNN6 genes but slightly decreased the content of the phosphoric acid is added mannose oligosaccharides compared to och1Δmnn1Δ and och1Δmnn1Δmnn4Δ strain did not show such a large difference. From these results, it was thought that genes other than the MNN4 and MNN6 genes were responsible for the addition of mannose phosphate.
실시예Example 3. 3. MNN4MNN4 와Wow MNN6MNN6 유전자와 Genes and 상동성이Homology 있는 there is S. S. cerevisiaecerevisiae 유전자 gene
N-당사슬 분석결과 MNN4와 MNN6 유전자가 추가로 결손된 och1Δmnn1Δmnn4Δ와 och1Δmnn1Δmnn4Δmnn6Δ 균주들에서도 만노스인산의 부가 활성이 완전히 제거 되지 않았기 때문에 만노스인산화 활성에 중요한 유전자들을 추가로 결손 하기 위한 연구를 수행하였다. N- sugar chain analysis result MNN4 and MNN6 Additionally the gene Missing In the och1Δmnn1Δmnn4Δ and och1Δmnn1Δmnn4Δmnn6Δ strains, since the addition activity of mannose phosphate was not completely eliminated, a study was conducted to further delete genes important for mannose phosphorylation activity.
이를 위해서 우선 MNN4와 MNN6 유전자들과 상동성이 있는 유전자들이 S. cerevisiae 효모에 추가로 있는지를 BLAST(basic local alignment tool)를 이용하여 미국 National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov)의 데이터 베이스를 탐색하였다. For this purpose, first, it is determined whether genes homologous to the MNN4 and MNN6 genes are additionally found in S. cerevisiae yeast using a basic local alignment tool (BLAST) in the US National Center for Biotechnology Information (http://www.ncbi.nlm). .nih.gov).
MNN4는 C-말단 부위에 KKKKEEEE의 반복 서열을 가지고 있어서 이 부위를 제외하고 BLAST를 하였고, 그 중 hypothetical protein YJR061W (NP_012595)를 선정하여 이를 MNN14로 명명하였다. Mnn14 단백질은 Mnn4와 마찬가지로 기질인 GDP-만노스와 결합에서 중요하다고 알려진 LicD 도메인을 가지고 있으나, Mnn4와는 다르게 C-말단에 KKKKEEEE의 반복 서열을 가지고 있지 않다. Mnn4와 Mnn14 단백질들 사이의 서열 동일성은 33%이고, 상동성은 44%로 계산되었다. MNN4 has a repeating sequence of KKKKEEEE at the C-terminal region, so BLAST was performed excluding this region. Among them, hypothetical protein YJR061W (NP_012595) was selected and named MNN14. Mnn14 protein, like Mnn4, has a LicD domain known to be important in binding to GDP-mannose, a substrate, but unlike Mnn4, it does not have a repeat sequence of KKKKEEEE at the C-terminus. The sequence identity between the Mnn4 and Mnn14 proteins was 33% and the homology was calculated as 44%.
한편, MNN6 유전자와 상동성이 있는 유전자들로는 KTR family에 속하는 YUR1, KTR2 , KTR4 , KTR5 및 KTR7 등이 검색되었다. Mnn6 단백질과 이들 단백질 사이의 동일성과 상동성은 도 4에 기재되어 있다.Meanwhile, YUR1 , KTR2 , KTR4 , KTR5, and KTR7 belonging to the KTR family were searched as genes homologous to the MNN6 gene. The identity and homology between the Mnn6 protein and these proteins are described in Figure 4.
실시예Example 4. 4. och1Δmnn1Δmnn4Δmnn6Δoch1Δmnn1Δmnn4Δmnn6Δ 균주에서 From strain MNN4MNN4 와Wow MNN6MNN6 상동 유전자들의 추가 결손 Additional deletion of homologous genes
실시예 1에서 제작한 och1Δmnn1Δmnn4Δmnn6Δ 균주로부터 만노스인산화와 관련이 되어 있을 것이라고 추정되는 MNN4와 MNN6의 상동 유전자 6종을 추가로 결손 하였다. From the och1Δmnn1Δmnn4Δmnn6Δ strain prepared in Example 1, six homologous genes of MNN4 and MNN6 , which are estimated to be related to mannose phosphorylation, were additionally deleted.
실시예 1에서와 동일한 방법으로 MNN14 , YUR1 , KTR2 , KTR4 , KTR5 및 KTR7 유전자들을 결손 하였다. 이를 위해서 loxP-URA3-loxP의 유전자 결손 카세트를 각각 6쌍의 프라이머 (MNN14_F/MNN14_R, Yur1_F/Yur1_R, Ktr2_F/Ktr2_R, Ktr4_F/Ktr4_R, Ktr5_F/Ktr5_R, Ktr7_F/Ktr7_R; 표 2 참조)들을 이용하여 중합효소 연쇄반응으로 1.7 kbp 절편을 얻었다 (도 5 (A)). MNN14 , YUR1 , KTR2 , KTR4 , KTR5 and KTR7 genes were deleted in the same manner as in Example 1. For this, the loxP-URA3-loxP gene deletion cassette was polymerized using 6 pairs of primers (MNN14_F/MNN14_R, Yur1_F/Yur1_R, Ktr2_F/Ktr2_R, Ktr4_F/Ktr4_R, Ktr5_F/Ktr5_R, Ktr7_F/Ktr7_R, see Table 2). A 1.7 kbp fragment was obtained by enzymatic chain reaction (Fig. 5 (A)).
이렇게 얻어진 유전자 파쇄 카세트들을 och1Δmnn1Δmnn4Δmnn6Δ 균주에 형질전환 방법으로 도입하였으며, 1 M 소르비톨이 첨가된 SC-URA 선택배지(1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base w/o amino acid, DO supplement -URA)에서 형질 전환체를 선별하였다. 그리고 선별된 형질 전환체들의 염색체 DNA를 추출하고 이를 주형으로 하여 Mnn14_CF/Ura3_CR, Yur1_CF/Ura3_CR, Ktr2_CF/Ura3_CR, Ktr4_CF/Ura3_CR, Ktr5_CF/Ura3_CR 및 Ktr7_CF/Ura3_CR 프라이머들을 이용한 중합효소 연쇄반응을 통하여 해당 유전자의 결손을 확인하였다(도5 (B)). The resulting gene disruption cassettes were introduced into the och1Δmnn1Δmnn4Δmnn6Δ strain by a transformation method, and 1 M sorbitol added SC-URA selective medium (1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base w/o amino acid, DO supplement- URA) was selected for transformants. Then, the chromosomal DNA of the selected transformants was extracted and used as a template for the polymerase chain reaction using Mnn14_CF/Ura3_CR, Yur1_CF/Ura3_CR, Ktr2_CF/Ura3_CR, Ktr4_CF/Ura3_CR, Ktr5_CF/Ura3_CR and Ktr7_CF/Ura3_CR primers. The defect of was confirmed (Fig. 5 (B)).
타깃 유전자의 결손을 위해서 들어간 선별용 마커 URA3는 실시예 1에서와 같이 Cre 단백질 발현을 위한 pSH68 벡터를 도입하고, 1 M 소르비톨이 들어간 SC-LEU 선택배지 (1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base w/o amino acid, DO supplement -LEU)에서 형질 전환체를 선별한 후, 이를 2% 포도당 대신 2% 갈락토오스가 첨가된 SC-LEU 선택배지 (1 M sorbitol, 2% galactose, 0.67% yeast nitrogen base w/o amino acid, DO supplement -LEU)에서 배양하여 Cre 단백질 발현 시켜서 제거하였다. 그리고 pSH68 벡터는 YPD 배지에서 연속 배양을 통해서 제거하였으며, 적절한 선별 배지에서 성장을 확인하여 URA3 마커가 제거된 5중 결손 균주 och1Δmnn1Δmnn4Δmnn6Δmnn14Δ (- mnn14Δ), och1Δmnn1Δmnn4Δmnn6Δyur1Δ (-yur1Δ), och1Δmnn1Δmnn4Δmnn6Δktr2Δ (- ktr2Δ), och1Δmnn1Δmnn4Δmnn6Δktr4Δ (- ktr4Δ), och1Δmnn1Δmnn4Δmnn6Δktr5Δ (- ktr5Δ), och1Δmnn1Δmnn4Δmnn6Δktr7Δ (- ktr7Δ)들을 제작하였다.As for the selection marker URA3, which was entered for the deletion of the target gene, as in Example 1, the pSH68 vector for Cre protein expression was introduced, and the SC-LEU selection medium containing 1 M sorbitol (1 M sorbitol, 2% glucose, 0.67% After selecting transformants in yeast nitrogen base w/o amino acid, DO supplement -LEU), they were prepared in SC-LEU selective medium (1 M sorbitol, 2% galactose, 0.67%) containing 2% galactose instead of 2% glucose. It was removed by culturing in yeast nitrogen base w/o amino acid, DO supplement -LEU) to express Cre protein. And the pSH68 vector was removed through continuous culture in YPD medium, and growth was confirmed in an appropriate selection medium to remove the URA3 marker, a five- fold deletion strain och1Δmnn1Δmnn4Δmnn6Δmnn14Δ (- mnn14Δ), och1Δmnn1Δmnn4Δmnn6Δyur1Δ ( -yur1Δ ), och1Δmnn1Δmnn4Δmnn6Δktr2Δ (- ktr2Δ), och1Δmnn1Δmnn4Δmnn6Δktr4Δ (- ktr4Δ), och1Δmnn1Δmnn4Δmnn6Δktr5Δ (- ktr5Δ), och1Δmnn1Δmnn4Δmnn6Δktr7Δ It was made of - (ktr7Δ).
실시예Example 5. 5. 만노스인산화Mannose phosphorylation 관련 유전자 다중 결손 균주들의 Of related gene multiple deletion strains NN -- 당사슬Our chain 구조 분석 Structure analysis
사중 결손 och1Δmnn1Δmnn4Δmnn6Δ 균주와 실시예 4에서 제작된 추가로 MNN4 또는 MNN6의 상동 유전자가 결손된 5중 결손 균주들(- mnn14Δ , - yur1Δ, -ktr2Δ, -ktr4Δ, -ktr5Δ, -ktr7Δ)의 N-당사슬을 비교 분석하였다. The quadruple deletion strain och1Δmnn1Δmnn4Δmnn6Δ as in Example 4, more homologous genes or MNN4 MNN6 as produced a defect of the defect in 5 strains oligosaccharide - N in the (- - mnn14Δ, yur1Δ, -ktr2Δ , -ktr4Δ, -ktr5Δ, -ktr7Δ) Were compared and analyzed.
우선, 1 M 소르비톨이 들어간 최소 배지 SC(1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base with amino acids)에서 28℃, 72시간 배양한 후에 그 세포를 수득하여 멸균수로 씻고, 실시예 2에서와 같은 방법으로 CWMs를 얻어서 그 N-당사슬을 분석하였다(도 6). MNN6의 상동 유전자가 결손 된 - yur1Δ, - ktr2Δ , -ktr4Δ, - ktr5Δ, - ktr7Δ 균주들은 och1Δmnn1Δmnn4Δmnn6Δ 균주와 마찬가지로 중성 당사슬(Man8GlcNAc2)과 함께 만노스인산이 부가된 (Man-P)2-Man8GlcNAc2과 Man-P-Man8GlcNAc2 당사슬 피크들이 검출되는 N-당사슬 프로파일을 보여주었다. First, the cells were obtained and washed with sterile water after culturing at 28° C. for 72 hours in a minimum medium SC (1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base with amino acids) containing 1 M sorbitol, and Example 2 CWMs were obtained in the same manner as in and the N -sugar chain was analyzed (FIG. 6). With a homologous gene of MNN6 defect - yur1Δ, - ktr2Δ, -ktr4Δ, - ktr5Δ, - ktr7Δ strains Like och1Δmnn1Δmnn4Δmnn6Δ strain neutral sugar chain (Man 8 GlcNAc 2) and a (P-Man) 2 -Man with mannose phosphate is added 8 GlcNAc 2 and Man-P-Man 8 GlcNAc 2 showed the N -sugar chain profile in which the oligosaccharide peaks were detected.
반면에 MNN4의 상동 유전자인 MNN14가 결손 된 - mnn14Δ 균주에서 만노스인산이 부가된 당사슬 피크들 없이 단일 Man8GlcNAc2 당사슬 피크를 보여주어 만노스인산 부가 능력이 상실 된 것을 알 수 있었다(도 6). On the other hand, with a homologous gene, the MNN4 MNN14 defect - mannose in mnn14Δ strain without the addition of phosphoric acid is oligosaccharide peaks single Man 8 GlcNAc 2 By showing the sugar chain peak, it was found that the ability to add mannose phosphate was lost (FIG. 6).
배양 환경이 영향을 미칠 수 있으므로 1 M 소르비톨이 첨가된 YPD 배지(1 M sorbitol, 2% galactose, 1% yeast extrat, 2% peptone)에서도 28℃, 72시간 배양한 후 상기와 같은 방법으로 CWMs의 N-당사슬을 분석하였다(도 7). 이 경우 MNN6의 상동 유전자가 결손 된 - yur1Δ, - ktr2Δ , - ktr4Δ, - ktr5Δ, - ktr7Δ 균주들은 och1Δmnn1Δmnn4Δmnn6Δ 균주와 동일하게 중성 당사슬과 함께 하나의 만노스인산이 부가된 Man-P-Man8GlcNAc2 당사슬 피크가 검출되었다. 그러나, MNN4의 상동 유전자인 MNN14가 결손 된 - mnn14Δ 균주에서는 Man8GlcNAc2 당사슬 피크 만이 검출되어 만노스인산 부가 능력이 상실된 것을 알 수 있었다(도 7). CWMs were cultured at 28°C for 72 hours in YPD medium (1 M sorbitol, 2% galactose, 1% yeast extrat, 2% peptone) added with 1 M sorbitol because the culture environment may have an effect. The N -sugar chain was analyzed (Fig. 7). In this case, the homologous gene of MNN6 defect - yur1Δ, - ktr2Δ, - ktr4Δ , - ktr5Δ, - ktr7Δ strains och1Δmnn1Δmnn4Δmnn6Δ strain in the same manner as one of the mannose phosphate is added with the neutral oligosaccharide Man-P-Man 8 GlcNAc 2 A sugar chain peak was detected. However, homologous genes, the MNN14 the loss of the MNN4 - the strain mnn14Δ Man 8 GlcNAc 2 It was found that only the oligosaccharide peak was detected and the ability to add mannose phosphate was lost (FIG. 7).
상기의 결과들은 배양 환경에 상관 없이, MNN14 유전자의 추가 결손을 통해서 S. cerevisiae 효모의 만노스인산화 활성을 완전히 제거할 수 있음을 보여준다. 이후 실험들은 모두 1 M 소르비톨이 들어간 최소 배지 SC (1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base with amino acids)에서 실험이 수행되었다. The above results show that, regardless of the culture environment, the mannose phosphorylation activity of S. cerevisiae yeast can be completely eliminated through the additional deletion of the MNN14 gene. All subsequent experiments were performed in a minimal medium SC (1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base with amino acids) containing 1 M sorbitol.
실시예Example 6. 6. och1Δmnn1Δmnn4Δmnn14Δoch1Δmnn1Δmnn4Δmnn14Δ 와Wow och1Δmnn1Δmnn14Δoch1Δmnn1Δmnn14Δ 균주의 제조Preparation of the strain
실시예 5에서 MNN14 유전자가 추가 결손 된 och1Δmnn1Δmnn4Δmnn6Δmnn14Δ 균주에서 만노스 인산화 능력이 완전히 제거된 것을 확인하였다. 이에 따라 MNN6 유전자가 결손 되지 않은 och1Δmnn1Δmnn4Δ 와 och1Δmnn1Δ 균주 등에서 MNN14 유전자의 추가 결손이 만노스인산화 능력에 어떠한 영향을 주는 지를 알아보고자 했다. The embodiments are MNN14 genes added in Example 5 defect och1Δmnn1Δmnn4Δmnn6Δmnn14Δ It was confirmed that the mannose phosphorylation ability was completely removed from the strain. Accordingly MNN6 Och1Δmnn1Δmnn4Δ and och1Δmnn1Δ with no gene deletion From strains, etc. We tried to find out how the additional deletion of the MNN14 gene affects the mannose phosphorylation ability.
이를 위해서 실시예 1에서 기술하고 제작한 och1Δmnn1Δmnn4Δ 와 och1Δmnn1Δ 균주들에 실시예 4에서 제작하였던 MNN14 유전자 결손 카세트를 도입하고 1 M sorbitol이 첨가된 SC-URA 선택배지에서 형질전환체를 선별하였다. 그리고 선별된 형질전환체들의 염색체 DNA를 추출하고 이를 주형으로 하여 Mnn14_CF와 Ura3_CR 프라이머들을 이용한 중합효소 연쇄반응을 통하여 MNN14 유전자의 결손을 확인하였다. MNN14 유전자의 결손을 위해서 들어간 선별용 마커 URA3는 실시예 1과 4에서와 같은 방법으로 제거하였으며, 마찬가지로 Cre 단백질 발현을 위한 pSH68 벡터도 YPD 배지에서 연속 배양을 통해서 제거하였다. To this end, och1Δmnn1Δmnn4Δ and och1Δmnn1Δ described and produced in Example 1 In the strains, the MNN14 gene deletion cassette prepared in Example 4 was introduced and transformants were selected in SC-URA selective medium to which 1 M sorbitol was added. Then, the chromosomal DNA of the selected transformants was extracted and the deletion of the MNN14 gene was confirmed through polymerase chain reaction using Mnn14_CF and Ura3_CR primers as a template. The selection marker URA3 entered for deletion of the MNN14 gene was removed in the same manner as in Examples 1 and 4, and the pSH68 vector for Cre protein expression was also removed through continuous culture in YPD medium.
또한, 상기 MNN4 및 MNN14 유전자들의 대표 결손 균주인 상기 och1Δmnn1Δmnn4Δmnn14Δ 균주를 och1Δmnn1Δmnn4Δmnn4paΔ로 명명하여 부타페스트 조약 하의 국제 기탁 기관인 한국 생명공학연구원에 2015년 4월 6일자로 기탁하여 KCTC12789P의 수탁번호를 부여받았다.In addition, the och1Δmnn1Δmnn4Δmnn14Δ , which is a representative deletion strain of the MNN4 and MNN14 genes The strain was named och1Δmnn1Δmnn4Δmnn4paΔ and was deposited with the Korea Research Institute of Bioscience and Biotechnology, an international depository under the Butafest Treaty, on April 6, 2015, and was given the accession number of KCTC12789P.
실시예Example 7. 7. och1Δmnn1Δmnn4Δmnn14Δoch1Δmnn1Δmnn4Δmnn14Δ 와Wow och1Δmnn1Δmnn14Δoch1Δmnn1Δmnn14Δ 균주의 Strain of NN -- 당사슬Our chain 분석 analysis
실시예 6에서 제작한 och1Δmnn1Δmnn4Δmnn14Δ와 och1Δmnn1Δmnn14Δ 균주들과 오중 결손 och1Δmnn1Δmnn4Δmnn6Δmnn14Δ 균주의 CWMs의 N-당사슬을 함께 분석하였다(도 8). Och1Δmnn1Δmnn4Δmnn14Δ and och1Δmnn1Δmnn14Δ prepared in Example 6 Strains and Faulty defect och1Δmnn1Δmnn4Δmnn6Δmnn14Δ The N -sugar chain of CWMs of the strain was analyzed together (FIG. 8).
N-당사슬의 분석은 실시예 2 및 5에서와 같이 1 M sorbitol이 들어간 SC 배지에서 28℃, 72시간 배양하여 얻은 세포들의 CWMs를 얻어서 실시하였다. Analysis of the N -sugar chain was carried out by obtaining CWMs of cells obtained by culturing at 28° C. for 72 hours in SC medium containing 1 M sorbitol as in Examples 2 and 5.
실험 결과 och1Δmnn1Δmnn4Δmnn6Δmnn14Δ와 och1Δmnn1Δmnn4Δmnn14Δ 균주는 만노스인산이 부가된 당사슬 없이 Man8GlcNAc2 당사슬의 단일 피크를 보여주었다(도 8). 반면에 och1Δmnn1Δmnn14Δ 균주는 Man8GlcNAc2 당사슬과 함께 만노스인산이 부가된 (Man-P)2-Man8GlcNAc2과 Man-P-Man8GlcNAc2 당사슬 피크들이 검출되는 프로파일을 보여주었다. 이러한 결과는 효모에서 만노스인산의 부가 활성을 제거하기 위해서는 MNN4와 MNN14 유전자가 모두 결손 되어야 한다는 것을 보여준다. Experimental results och1Δmnn1Δmnn4Δmnn6Δmnn14Δ and och1Δmnn1Δmnn4Δmnn14Δ The strain is Man 8 GlcNAc 2 without mannose phosphate added sugar chain It showed a single peak of the sugar chain (Fig. 8). Whereas och1Δmnn1Δmnn14Δ The strains were Man 8 GlcNAc 2 sugar chain and mannose phosphate added (Man-P) 2 -Man 8 GlcNAc 2 and Man-P-Man 8 GlcNAc 2 The profile in which oligosaccharide peaks were detected was shown. These results show that both MNN4 and MNN14 genes must be deleted in order to remove the additive activity of mannose phosphate in yeast.
실시예Example 8. 8. MNN4MNN4 와Wow MNN14MNN14 유전자의 complementation 실험 Gene complementation experiment
만노스인산화 활성의 제거가 확인된 두 균주 och1Δmnn1Δmnn4Δmnn14Δ와 och1Δmnn1Δmnn4Δmnn6Δmnn14Δ에 MNN4 또는 MNN14 유전자를 발현하여 만노스인산화 능력의 회복 여부를 보는 complementation 실험을 수행하였다.Complementation experiments were performed to see whether the mannose phosphorylation ability was recovered by expressing the MNN4 or MNN14 gene in two strains och1Δmnn1Δmnn4Δmnn14Δ and och1Δmnn1Δmnn4Δmnn6Δmnn14Δ in which the removal of mannose phosphorylation activity was confirmed.
8-1: YEp352-GAP, YEp352-Mnn4, 및 YEp352-Mnn14 벡터의 제작8-1: Construction of YEp352-GAP, YEp352-Mnn4, and YEp352-Mnn14 vectors
먼저, Mnn4 단백질 발현을 위한 YEp352-Mnn4와 control 벡터인 YEp352-GAP 벡터를 하기와 같이 제작하였다. First, YEp352-Mnn4 for expression of Mnn4 protein and YEp352-GAP vector as a control vector were constructed as follows.
구체적으로, S. cerevisiae의 GAPDH 프로모터(0.8 kb) 및 터미네이터(0.2 kb)는 L3262 균주의 염색체 DNA로부터 GAPDHp-F1/GAPDHp-R1와 GAPDHt-F1/GAPDHt-R1 프라이머들을 이용하여 중합효소 연쇄반응을 통해서 증폭하였다. Specifically, the GAPDH promoter (0.8 kb) and terminator (0.2 kb) of S. cerevisiae performed polymerase chain reaction using GAPDHp-F1/GAPDHp-R1 and GAPDHt-F1/GAPDHt-R1 primers from the chromosomal DNA of the L3262 strain. It was amplified through.
증폭된 DNA 절편은 pDrive 벡터(QIAGEN, Germany)에 각각 삽입하여 pDrive-GAPDHp와 pDrive-GAPDHt 벡터들을 제작하였다. 재조합효소 EcoRI과 KpnI을 사용하여 pDrive-GAPDHp으로부터 GAPDH 프로모터를 절단하였으며, 같은 재조합 효소로 절단된 YEp352 벡터에 삽입하여 YEp352-GAPDHp 벡터를 제작하였다. 다음으로 KpnI과 PstI 효소를 사용하여 pDrive-GAPDHt로부터 GAPDH 터미네이터를 절단하여 같은 재조합 효소로 절단된 YEp352-GAPDHp 벡터에 삽입하여 YEp352-GAP 벡터를 제작하였다. The amplified DNA fragments were respectively inserted into pDrive vectors (QIAGEN, Germany) to produce pDrive-GAPDHp and pDrive-GAPDHt vectors. Using recombinant enzyme EcoR I and Kpn I cut was the GAPDH promoter from pDrive-GAPDHp, and then inserted into a vector digested with the same YEp352 recombinant enzyme was produced in the YEp352-GAPDHp vector. Next, inserted on the Kpn I and YEp352-GAPDHp vector digested with the Pst I enzyme from pDrive-GAPDHt a recombinase, such as by cutting the GAPDH terminator was produced in the YEp352-GAP vector.
MNN4 유전자는 S. cerevisiae BY4741 균주의 염색체 DNA로부터 Mnn4_F와 Mnn4_R 프라이머를 이용하여 증폭하였으며, 제한효소 BamHI와 SpeI를 처리한 절편을 BamHI와 XbaI을 처리한 YEp352-GAP 벡터에 삽입하여 YEp352-Mnn4 벡터를 제작하였다. The MNN4 gene was amplified from chromosomal DNA of S. cerevisiae BY4741 strain using Mnn4_F and Mnn4_R primers, and the fragments treated with restriction enzymes BamH I and Spe I were inserted into the YEp352-GAP vector treated with BamH I and Xba I, and YEp352 -Mnn4 vector was constructed.
Mnn14 단백질 발현을 위한 YEp352-Mnn14 벡터 제작을 위해서 MNN14 유전자(2.8 kb)는 S. cerevisiae L3262의 염색체 DNA로부터 Y-Mnn14-F와 Y-Mnn14-R 프라이머를 이용한 중합효소 연쇄반응으로 증폭하였다. 여기에 KpnI 제한 효소를 처리하여 절단한 단편을 YEp352-GAP 벡터의 GAPDH 프로모터 뒤의 KpnI 위치에 삽입하여 YEp352-MNN14 벡터를 제작하였다.For the YEp352-Mnn14 vector construction for expression MNN14 Mnn14 protein gene (2.8 kb) it was amplified by polymerase chain reaction using the chromosomal DNA from the F-Y-Mnn14 and Mnn14-Y-R primer of S. cerevisiae L3262. Here, a fragment cut by treatment with a KpnI restriction enzyme was inserted into the KpnI position after the GAPDH promoter of the YEp352-GAP vector, thereby constructing the YEp352-MNN14 vector.
상기 벡터 제작에 사용한 프라이머의 서열은 하기와 같았다. The sequence of the primers used to construct the vector was as follows.
8-2: och1Δmnn1Δmnn4Δmnn14Δ 균주의 만노스인산화 회복 실험8-2: och1Δmnn1Δmnn4Δmnn14Δ Mannose phosphorylation recovery experiment of strain
만노스인산화 능력이 없는 och1Δmnn1Δmnn4Δmnn14Δ 균주에 실시예 8-1의 YEp352-GAP, YEp352-Mnn4와 YEp352-Mnn14 벡터를 통상적인 형질전환 방법으로 도입하였다. 그리고 이를 실시예 2 및 5에서와 같이 1 M 소르비톨이 들어간 SC-Ura3 배지에서 28℃, 72시간 배양하여 얻은 세포들의 CWMs를 얻어서 N-당사슬을 분석하였다(도 9). 그 결과 Mnn4 또는 Mnn14 단백질을 발현한 경우 모두에서 만노스인산화 능력이 회복되었다. 그러나 Mnn4를 발현한 경우에 비해서 Mnn14를 발현했을 때 만노스인산의 부가 능력이 훨씬 높아서 Mnn4보다 Mnn14가 만노스인산화에 있어서 보다 핵심적인 역할을 수행한다는 것을 추정할 수 있었다. Och1Δmnn1Δmnn4Δmnn14Δ without mannose phosphorylation ability The YEp352-GAP, YEp352-Mnn4 and YEp352-Mnn14 vectors of Example 8-1 were introduced into the strain by a conventional transformation method. In addition, as in Examples 2 and 5, CWMs of the cells obtained by culturing them at 28° C. for 72 hours in SC-Ura3 medium containing 1 M sorbitol were obtained, and the N -sugar chain was analyzed (FIG. 9). As a result, the mannose phosphorylation ability was restored in all cases where Mnn4 or Mnn14 protein was expressed. However, compared to the case where Mnn4 was expressed, when Mnn14 was expressed, the additional ability of mannose phosphate was much higher, so it could be estimated that Mnn14 played a more important role in mannose phosphorylation than Mnn4.
8-3: och1Δmnn1Δmnn4Δmnn6Δmnn14Δ 균주의 만노스인산화 회복 실험8-3: och1Δmnn1Δmnn4Δmnn6Δmnn14Δ Mannose phosphorylation recovery experiment of strain
실시예 8-2에서와 같이 만노스인산화 능력이 없는 och1Δmnn1Δmnn4Δmnn6Δmnn14Δ 균주에 YEp352-GAP, YEp352-Mnn4와 YEp352-Mnn14 벡터를 통상적인 형질전환 방법으로 도입하고, 그 세포들의 CWMs를 얻어서 N-당사슬을 분석하였다(도 10). 그 결과 실시예 8-2에서와 마찬가지로 Mnn4 또는 Mnn14 단백질을 발현한 경우 만노스인산화 능력이 회복되었으며, Mnn4에 비해서 Mnn14를 발현했을 때 만노스인산화 능력이 훨씬 높아졌다. 이러한 결과들로부터 Mnn14가 Mnn4에 비해서 만노스인산의 부가에서 보다 핵심적인 역할을 수행한다는 것을 추정할 수 있었다. Och1Δmnn1Δmnn4Δmnn6Δmnn14Δ without mannose phosphorylation ability as in Example 8-2 YEp352-GAP, YEp352-Mnn4 and YEp352-Mnn14 vectors were introduced into the strain by a conventional transformation method, and CWMs of the cells were obtained, and the N -sugar chain was analyzed (FIG. 10). As a result, as in Example 8-2, when the Mnn4 or Mnn14 protein was expressed, the mannose phosphorylation ability was restored, and the mannose phosphorylation ability was much higher when Mnn14 was expressed compared to Mnn4. From these results, it could be estimated that Mnn14 plays a more important role in the addition of mannose phosphate compared to Mnn4.
실시예Example 9. 9. MNN4MNN4 와Wow MNN14MNN14 유전자 결손에 따른 분비 발현 단백질의 Of secreted-expressed proteins according to gene deletion NN -- 당사슬의Our chain 변화 분석 Change analysis
상기의 실시예 2 내지 8에서는 효모의 세포 벽 만노단백질(cell wall mannoproteins, CWMs)의 N-당사슬을 분석한 것으로, 추가 확인을 위해서 이후의 실시예에서는 효모로부터 분비 발현되는 당단백질의 N-당사슬을 분석하는 실험을 수행하였다.In Examples 2 to 8 above, the N -sugar chain of yeast cell wall mannoproteins (CWMs) was analyzed, and for further confirmation, the N -sugar chain of the glycoprotein secreted and expressed from yeast in the following examples. An experiment was performed to analyze.
9-1: Gas1 단백질 분비 발현 벡터 제작9-1: Gas1 protein secretion expression vector construction
모델 당단백질로서 S. cerevisiae 효모의 Gas1 단백질을 선택하고, 분비 발현을 위하여 C-말단의 glycosylphosphatidylinositol (GPI)-anchoring motif가 제거된 1 - 490번 아미노산 잔기의 단백질을 발현하는 유전자를 아래와 같은 중합효소 연쇄반응을 이용하여 증폭하였다(Gil et al, J Biotech 2015). S. cerevisiae L3262 균주의 genomic DNA를 추출한 후 이를 주형으로 하고 p-Gas1-F와 p-Gas1-R-1 프라이머를 사용하여 첫 번째 중합효소 연쇄반응으로 증폭하여 DNA 절편을 얻었다. 그리고 이를 다시 주형으로 하여 pGas1-F와 p-Gas1-R-2 프라이머를 이용하여 중합효소 연쇄반응으로 증폭하여 C-말단에 정제를 위한 8개의 His 잔기로 이루어진 His-tag을 첨가된 DNA 절편을 얻었다. 상기 증폭된 DNA는 DNA2.0 (Menlo Park, CA, 미국)의 pD1211 벡터에 Electra 방법(DNA2.0)으로 제조사에서 제공하는 프로토콜에 따라서 클로닝하였으며, 최종 제작된 벡터는 pD1211-Gas1p로 명명하였다. As the model glycoprotein, the Gas1 protein of S. cerevisiae yeast was selected, and the gene expressing the protein of amino acids 1-490 from which the C-terminal glycosylphosphatidylinositol (GPI)-anchoring motif was removed for secretion expression was polymerized as follows. It was amplified using a chain reaction (Gil et al, J Biotech 2015). After extracting genomic DNA of S. cerevisiae L3262 strain, it was used as a template and amplified by the first polymerase chain reaction using primers p-Gas1-F and p-Gas1-R-1 to obtain DNA fragments. Then, using the primers pGas1-F and p-Gas1-R-2 as a template, amplified by polymerase chain reaction, and a DNA fragment containing a His-tag consisting of 8 His residues for purification at the C-terminus was added. Got it. The amplified DNA was cloned into a pD1211 vector of DNA2.0 (Menlo Park, CA, USA) according to the protocol provided by the manufacturer by the Electra method (DNA2.0), and the final constructed vector was named pD1211-Gas1p.
상기 벡터 제작에 사용한 프라이머의 서열들은 하기와 같다. The sequences of the primers used to construct the vector are as follows.
9-2: 재조합 Gas1 단백질의 분비 발현 및 정제9-2: Secretion expression and purification of recombinant Gas1 protein
상기에서 제작된 pD1211-Gas1p 벡터를 실시예 1에서 제작한 och1Δmnn1Δmnn4Δmnn6Δ 균주와 실시예 6에서 제작한 och1Δmnn1Δmnn4Δmnn14Δ 균주에 통상적인 형질전환 방법으로 도입하고, 1 M 소르비톨이 첨가된 SC-URA 선택배지(1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base w/o amino acid, DO supplement -URA)에서 선별하여 재조합 Gas1 단백질을 분비 발현하는 균주들을 제작하였다. 또한, och1Δmnn1Δmnn4Δmnn14Δ 균주에 실시예 8에서 제작한 YEp352-Mnn4와 YEp352-Mnn14 벡터와 함께 pD1211-Gas1p 벡터를 통상적인 형질전환 방법으로 도입하고 1 M 소르비톨이 첨가된 SC-URA, LEU 선택배지에서 선별하여 Gas1을 분비 발현하는 och1Δmnn1Δmnn4Δmnn14Δ/Mnn4/Gas1와 och1Δmnn1Δmnn4Δmnn14Δ/Mnn14/Gas1 균주들을 제작하였다. Produced in the pD1211-Gas1p vector for Example 1, och1Δmnn1Δmnn4Δmnn6Δ strain as in Example 6 and was introduced by a conventional transformation method to och1Δmnn1Δmnn4Δmnn14Δ strain produced in, 1 M sorbitol is added SC-URA selection media (prepared in 1 M sorbitol, 2% glucose, 0.67% yeast nitrogen base w/o amino acid, DO supplement -URA) to produce strains secreting and expressing the recombinant Gas1 protein. Also, och1Δmnn1Δmnn4Δmnn14Δ Introduce the pD1211-Gas1p vector together with the YEp352-Mnn4 and YEp352-Mnn14 vectors produced in Example 8 to the strain by a conventional transformation method, and secrete Gas1 by selecting in SC-URA and LEU selective media to which 1 M sorbitol was added. Expressing och1Δmnn1Δmnn4Δmnn14Δ /Mnn4/Gas1 and och1Δmnn1Δmnn4Δmnn14Δ /Mnn14/Gas1 strains were prepared.
상기에서 제작한 4종의 재조합 Gas1 분비 발현 효모 균주들을 1 M 소르비톨이 첨가된 적절한 선택배지 (SC-URA 또는 SC-URA, LEU)를 사용하여 28℃ 에서 3일 동안 배양하였다. 그리고 이를 통해서 얻은 배양 상등액을 통상의 His-tag 친화 컬럼을 사용하여 정제하였다(Gil et al, J Biotech 2015).Four types of recombinant Gas1 secretion-expressing yeast strains prepared above were cultured at 28°C for 3 days using an appropriate selective medium (SC-URA or SC-URA, LEU) to which 1 M sorbitol was added. And the culture supernatant obtained through this was purified using a conventional His-tag affinity column (Gil et al, J Biotech 2015).
9-3: 정제된 Gas1 단백질의 N-당사슬 분석 9-3: Analysis of N -sugar chain of purified Gas1 protein
상기의 실험을 통해서 정제한 Gas1 단백질들의 N-당사슬은 기존 문헌(Lee KJ et al, 2013, Glycoconj J)에 기술 된 바와 같이 분리 정제하였다. 간략히 기술하면, 10 mg의 정제된 Gas1 단백질을 denaturation하고 직접 PNGase F (New Englad Biolabs)를 처리하여 당사슬을 유리한 후에 graphitized carbon 컬럼(Alltech, Lexington, MA, USA)을 이용한 solid phase extraction 방법을 이용하여 정제하였다. 이렇게 정제한 N-당사슬들은 실시예 2의 DNA 시퀀서를 이용한 방법으로 분석하였다(도 11). The N -sugar chain of Gas1 proteins purified through the above experiment was separated and purified as described in the existing literature (Lee KJ et al, 2013, Glycoconj J). Briefly, after denaturation of 10 mg of purified Gas1 protein and directly treatment with PNGase F (New Englad Biolabs) to liberate sugar chains, a solid phase extraction method using a graphitized carbon column (Alltech, Lexington, MA, USA) was used. Purified. The thus-purified N -sugar chains were analyzed by the method using the DNA sequencer of Example 2 (FIG. 11).
분석 결과 och1Δmnn1Δmnn4Δmnn6Δ 균주에서 분비 발현한 재조합 Gas1 단백질에는 만노스인산이 부가된 당사슬들이 많은 양 부착되는 데 반하여, och1Δmnn1Δmnn4Δmnn14Δ 균주에서 분비 발현한 재조합 Gas1 단백질에는 만노스인산이 부가된 당사슬들이 관찰되지 않았다(도 11의 두 번째와 세 번째 프로파일). 이는 실시예 5와 7에서 효모의 CWM의 N-당사슬을 이용하여 분석한 결과들과(도 6과 8) 일치하며, S. cerevisiae 효모에서 분비 발현하는 당단백질의 당사슬에 만노스인산이 부가되지 않게 하기 위해서는 MNN4와 MNN14 유전자를 동시에 결손 해야 한다는 것을 확인해준다. 이러한 사실은 och1Δmnn1Δmnn4Δmnn14Δ 균주에 Mnn4 또는 Mnn14 단백질을 발현하여 complementation한 균주들(och1Δmnn1Δmnn4Δmnn14Δ/Mnn4와 och1Δmnn1Δmnn4Δmnn14Δ/Mnn14)에서 재조합 Gas1 단백질을 분비 발현한 실험을 통해서 더욱 확실하게 확인할 수 있다(도 11의 네 번째와 다섯 번째 프로파일). 두 경우 모두 만노스인산의 부가 능력이 회복되는 것을 확인할 수 있다. 이 경우에 주목 할 점은 Mnn4를 och1Δmnn1Δmnn4Δmnn14Δ 균주에서 발현 시켰을 때에는 만노스인산이 하나 부가된 당사슬만이 적은 양 검출되는 데 반하여, Mnn14를 발현 시켰을 때 만노스인산이 하나 부가된 당사슬 peak의 세기가 증가할 뿐 만 아니라 두 개가 부가된 당사슬에 매우 강한 세기의 peak로 검출되었다는 것이다. 이는 Mnn14의 만노스인산 부가 능력이 Mnn4보다 훨씬 우수하다는 것을 보여준다.Analysis result och1Δmnn1Δmnn4Δmnn6Δ While a large amount of mannose phosphate-added sugar chains are attached to the recombinant Gas1 protein secreted and expressed in the strain, och1Δmnn1Δmnn4Δmnn14Δ In the recombinant Gas1 protein secreted and expressed in the strain, oligosaccharides to which mannose phosphate was added were not observed (second and third profiles in FIG. 11). This is consistent with the results analyzed using the N -sugar chain of the yeast CWM in Examples 5 and 7 (Figs. 6 and 8), so that mannose phosphate was not added to the sugar chain of the glycoprotein secreted and expressed in S. cerevisiae yeast. In order to do this, it confirms that the MNN4 and MNN14 genes must be deleted at the same time. This fact is och1Δmnn1Δmnn4Δmnn14Δ The recombinant Gas1 protein was secreted and expressed in strains complemented by expressing Mnn4 or Mnn14 protein in the strain ( och1Δmnn1Δmnn4Δmnn14Δ /Mnn4 and och1Δmnn1Δmnn4Δmnn14Δ /Mnn14). . In both cases, it can be seen that the additional ability of mannose phosphate is restored. In this case, it should be noted that when Mnn4 was expressed in the och1Δmnn1Δmnn4Δmnn14Δ strain, only a small amount of the sugar chain with one mannose phosphate was detected, whereas when Mnn14 was expressed, the intensity of the peak of the sugar chain with one mannose phosphate added only increased. In addition, it was detected as a very strong peak in the added sugar chain. This shows that Mnn14's ability to add mannose phosphate is much better than that of Mnn4.
9-4: 정제된 Gas1 단백질의 등전점 전기영동9-4: Isoelectric point electrophoresis of purified Gas1 protein
상기의 정제된 Gas1 단백질들의 고유 등전점(isoelctric point)을 통상의 등전점 전기영동(isoelctric focusing: IEF) 방법을 이용하여 분석하였다. 간략히 기술하면 IEF 겔(ThermoFisher Scientific, Waltham, MA, USA)을 cathod와 anode 버퍼(ThermoFisher Scientific)로 채워진 챔버(ThermoFisher Scientific)에 장착하고, sample 버퍼(ThermoFisher Scientific)가 첨가된 Gas1 단백질 시료를 IEF 겔의 well에 로딩한 후 제조사에서 제공하는 프로토콜을 따라서 전기영동을 수행하였다. 전기영동이 끝난 겔은 fixation 버퍼(12% Trichloroacetic acid)에 30분 동안 반응 한 후, 쿠마시블루 염색을 진행하여 그 결과를 확인하였다(도 12). The intrinsic isoelctric point of the purified Gas1 proteins was analyzed using a conventional isoelctric focusing (IEF) method. Briefly, an IEF gel (ThermoFisher Scientific, Waltham, MA, USA) was mounted in a chamber filled with cathod and anode buffer (ThermoFisher Scientific) (ThermoFisher Scientific), and a Gas1 protein sample added with the sample buffer (ThermoFisher Scientific) was added to the IEF gel. After loading into the well of, electrophoresis was performed according to the protocol provided by the manufacturer. The gel after electrophoresis was reacted in fixation buffer (12% Trichloroacetic acid) for 30 minutes, and then coomassie blue staining was performed to confirm the result (FIG. 12).
만노스인산은 음 전하를 띄고 있어서 만노스인산이 부가될 수록 단백질의 등전점은 더 낮아진다. 따라서 och1Δmnn1Δmnn4Δmnn6Δ 균주에서 분비 발현한 재조합 Gas1 단백질은 만노스인산이 많이 부가되어서 주요 단백질 band의 등전점이 pH 3 이하에 위치하며 끌리듯이 나타났다(도 12의 첫 번째 lane). 반면에, och1Δmnn1Δmnn4Δmnn14Δ 균주에서 분비 발현한 재조합 Gas1 단백질은 주요 band가 pH 4 이상에서 관찰되어(도 12의 두 번째 lane) 등전점이 높아지는 결과를 얻어서, 도 11의 N-당사슬 분석에서 만노스인산의 부가 능력이 제거된 것과 일치하는 결과를 얻었다. 또한 Mnn4 또는 Mnn14 단백질 발현을 complementation한 균주들(och1Δmnn1Δmnn4Δmnn14Δ/Mnn4와 och1Δmnn1Δmnn4Δmnn14Δ/Mnn14)에서 분비 발현한 재조합 Gas1 단백질들은 och1Δmnn1Δmnn4Δmnn14Δ 균주에서 분비 발현한 단백질에 비해서 등전점이 확연히 낮아지는 것을 확인할 수 있었다(도 12의 세 번째와 네 번째 lane). 여기서도 Mnn14 단백질 발현을 complementation한 균주가 Mnn4 complementation 균주에 비해서 등전점이 더 낮아지는 것으로부터 Mnn14의 만노스인산 부가 능력이 보다 우수함을 확인할 수 있었다. Since mannose phosphate has a negative charge, the more mannose phosphate is added, the lower the isoelectric point of the protein. So och1Δmnn1Δmnn4Δmnn6Δ Recombinant Gas1 protein secreted and expressed in the strain showed a lot of addition of mannose phosphate, so that the isoelectric point of the main protein band was located at
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.
<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> An essential gene MNN14 for mannosylphosphorylation in Saccharomyces cerevisiae and a method for producing recombinant glycoprotein using a MNN14-defected microorganism <130> KPA150188-KR-P1-D1 <150> 10-2015-0064855 <151> 2015-05-08 <160> 49 <170> KopatentIn 2.0 <210> 1 <211> 935 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Met Met Leu Ser Leu Arg Arg Phe Ser Met Tyr Val Leu Arg Ser Leu 1 5 10 15 Arg Leu His Phe Lys Lys Ile Ile Ile Thr Leu Leu Thr Ile Gln Leu 20 25 30 Leu Phe Ile Thr Ile Phe Val Leu Gly Gly Arg Ser Ser Ile Ile Asp 35 40 45 Gly Asn Trp Lys Ser Phe Met Ala Leu Phe Phe Lys Pro Leu Ala Tyr 50 55 60 Thr Asn Arg Asn Asn Asn His Ala Ser Phe Asp Leu Arg Ser Lys Asp 65 70 75 80 Asn Val Ala Lys Leu Tyr Glu Lys Met Asn Phe Asp Thr Ser Gly Lys 85 90 95 Trp Ile Asp Thr Tyr Thr Leu Lys Asn Asn Leu Leu Thr Val Lys Met 100 105 110 Gly Pro Glu Lys Gly Gln Val Leu Asp Ser Val Asp Glu Leu Arg Tyr 115 120 125 Tyr Asp Asn Asp Pro Arg Leu Val Trp Ser Val Leu Leu Asp His Leu 130 135 140 Leu Glu Ser Asp Ser Asn Glu Tyr Ala Phe Ser Trp Tyr Asp Trp Ala 145 150 155 160 Asn Phe Asp Ser Thr Asn Lys Leu Ile Ala Leu Arg His Thr Asn Ile 165 170 175 Ser Cys Gln Phe Val Cys Glu Gly Ala Phe Asp Lys Asn Val Leu Glu 180 185 190 Met Val Glu Ser Glu Val Gln Glu Pro Leu Phe Val Thr Asn Arg Asn 195 200 205 Lys Tyr Asp Glu Ser Leu Trp Tyr Asn Arg Val Arg Lys Val Val Asp 210 215 220 Ser Asn Ser Val Gln Gln Ala Ile His Asp His Cys Met Asn Asn Asp 225 230 235 240 Ala Tyr Ser Asn Gly Thr Pro Phe Glu Leu Pro Phe Ile Ile Ser Glu 245 250 255 Ile Ser Glu Arg Leu Arg Pro Glu Val Tyr Asp Leu Gln Ala Lys Asn 260 265 270 His Leu Leu Tyr Ser Asn Phe Thr Pro Leu Ser Leu Thr Val Leu Asp 275 280 285 Ser Asp Lys Asp Ala Tyr Arg Ile Asn Leu Lys Thr Thr Asp Ser Ser 290 295 300 Lys Ser Asn Ile Val Gln Thr Asn Leu Leu Gln Asn Tyr Ile Lys Arg 305 310 315 320 His Arg Asn Glu Met Val Asn Gly Asp Leu Ile Phe Asn His Thr Ser 325 330 335 Met Phe Glu Lys Phe Leu His His Gly Ser Thr Lys Lys Arg Lys Leu 340 345 350 Asp Val Glu Ala Leu Asp Lys Thr Ile Tyr Ala Gly Glu Tyr Leu Glu 355 360 365 Leu Ser Pro Ser Asp Phe Gln Phe Asn Ala Lys Glu Arg Ile Ile Glu 370 375 380 Leu Glu Thr Arg Leu Arg Ser Glu Gly Leu Pro Ser His Asp Thr His 385 390 395 400 Tyr Leu Arg Ser Leu Lys Thr Ser Val Asn Thr Ser Pro Ala Leu Gln 405 410 415 Gln Lys Tyr Phe Ala Glu Ala Ser Asp Ile Thr Asp Ala Thr Ala Asp 420 425 430 Gly His His Arg Asp Arg Arg Phe Phe Ser Ile Gly His Asn Leu Leu 435 440 445 Asn Asp Pro Gln Glu Phe Glu Ala Arg Leu Asn Ser Leu Ile Arg Asn 450 455 460 Phe Gln Lys Phe Val Lys Ala Asn Gly Leu Ile Ser Trp Leu Ser His 465 470 475 480 Gly Thr Leu Tyr Gly Tyr Leu Tyr Asp Gly Leu Lys Phe Pro Trp Asp 485 490 495 Val Asp His Asp Leu Gln Met Pro Ile Lys His Leu His Tyr Leu Ser 500 505 510 Gln Tyr Phe Asn Gln Ser Leu Ile Leu Glu Asp Pro Arg Glu Gly Asn 515 520 525 Gly Arg Phe Leu Leu Asp Val Gly Ser Ala Ile Thr Val Gly Val His 530 535 540 Gly Asn Gly Glu Asn Asn Ile Asp Ala Arg Phe Ile Asp Ile Asp Ser 545 550 555 560 Gly Ile Tyr Ile Asp Ile Thr Gly Leu Ser Val Ser Ser Asp Ala Ala 565 570 575 Lys Gln Tyr Met Ser Lys Phe Val Glu Glu Glu Ser Ser Gly Glu Ser 580 585 590 Phe Ser Ala Leu Ile Glu Asp Tyr Lys Phe Asp Glu Asn Asp Tyr Phe 595 600 605 Asp Glu Val Asp Gly Arg Glu Gly Leu Ala Lys Tyr Thr Ile His Glu 610 615 620 Leu Met Glu Trp Val Asn Ser His Pro Asp Asp Phe Thr Asp Ala Glu 625 630 635 640 Lys Asn Leu Val Thr Lys Thr Tyr Lys Lys Glu Leu Ala Ile Ser Arg 645 650 655 Ser Asp Tyr Ala Glu Lys Asp Leu Ser Pro Lys Gln Arg Tyr Leu Val 660 665 670 Asn Glu Lys Tyr Asn Leu Tyr Asn Cys Arg Asn Gln His Phe Ser Ser 675 680 685 Leu Asn Ile Ile Ser Pro Leu Arg Asn Thr Met Phe Ser Gly Val Ser 690 695 700 Ala Phe Val Pro Asn Arg Pro Ile Ala Thr Leu Asn Asn Glu Tyr Lys 705 710 715 720 Val Pro Ala Lys Tyr Gly Leu Leu Ser Phe Gln Gly Lys Val Tyr Leu 725 730 735 Pro Glu Phe Arg Tyr Trp Phe Ser Phe Ala Asp Met Lys Lys Phe Ala 740 745 750 Asn Leu Gln Leu Lys Glu Pro Lys Ile Thr Arg Leu Glu Ser Pro Leu 755 760 765 Asn Asp Leu Lys Phe Ser Asp Ile Ser Leu Leu Ile Thr Asn Ile Leu 770 775 780 Lys Cys Gly Phe His Ser Val Phe Ala Ser Leu Phe Asn Ser Phe Asp 785 790 795 800 Ser Thr Val Tyr Arg Leu Lys Glu Leu Glu Ile Gln Tyr Asp Pro Ser 805 810 815 Leu Ser Glu Glu Glu Lys Ser Ser Leu Leu Lys Thr Leu Arg Arg Gly 820 825 830 Met Ser Lys Lys Ile Lys Ser Pro Glu Lys Asp Pro Ile Ile Tyr Ile 835 840 845 Tyr Glu Arg Lys Leu Trp Glu Asn Val Glu Lys Leu Leu Asn Ala Ser 850 855 860 Asn Ile Tyr Asn Ile Ala Ser Gln Val Glu Lys Glu Lys Gly Lys Glu 865 870 875 880 Phe Val Glu Arg Ser Gln Gln Val Tyr Glu Arg Asn Phe Asp Gly Phe 885 890 895 Arg Leu Pro Asp Gly Gly Asn Ser Lys Thr Val Asn Asp Leu Asn Ser 900 905 910 Lys Gly Leu Asn Leu Phe Gly Asp Asn Lys Lys Thr Ser Asn Asn Ile 915 920 925 Phe Gly Ser Asp Gln Lys Tyr 930 935 <210> 2 <211> 2808 <212> DNA <213> Saccharomyces cerevisiae <400> 2 atgatgttat cactgcgcag gttctccatg tacgttttga gatctctgcg gcttcacttt 60 aaaaagataa tcattactct tctaactatc cagttactat tcattaccat atttgtattg 120 ggcggtcgct cgtcgattat tgacggtaac tggaagtcat tcatggcgct ctttttcaaa 180 ccgcttgctt acactaacag aaacaacaac catgcttctt tcgatctgag atcaaaagac 240 aacgtagcca aactttacga aaaaatgaat tttgatactt caggcaaatg gatcgacacg 300 tacaccttga agaataatct tctcactgtg aaaatgggtc ctgaaaaagg gcaagttctt 360 gattcggtag atgaattgag atattacgat aacgacccaa ggctggtatg gtcagtttta 420 ctagatcact tattagaatc agattccaat gaatacgcat tttcgtggta cgattgggct 480 aattttgact ctacaaacaa actcattgca ctgagacaca cgaacatatc ttgccagttc 540 gtttgcgagg gtgcctttga taaaaatgtg ctagaaatgg tagagagtga agtccaagag 600 cctttattcg tcacaaatag gaataaatat gacgaatcgc tctggtacaa cagggtaaga 660 aaggttgtcg attctaattc tgtgcagcaa gccatacatg atcactgcat gaataatgac 720 gcgtattcca atggtactcc cttcgaattg ccttttatca taagcgaaat ttctgaaagg 780 ttgaggccag aagtgtatga cttacaagcc aaaaaccact tgttatattc taactttact 840 ccactgtcat taaccgtact ggacagcgat aaagatgcat acagaatcaa tttgaagaca 900 acagactctt ccaaatcaaa tatagtacag acaaatctac tacagaatta cattaagagg 960 cacagaaatg aaatggtaaa tggcgacctc attttcaacc acacttccat gtttgaaaaa 1020 tttttacatc atggatccac taaaaaaagg aaacttgacg ttgaagcgtt ggataaaaca 1080 atatacgctg gagagtatct agaactatca ccatctgatt tccaattcaa tgcaaaagag 1140 aggatcattg aattagagac caggctcagg tctgaaggcc taccatctca tgatacccac 1200 tatttacgaa gtttaaagac gtccgtaaat acgtcccctg cattacagca aaagtatttc 1260 gcagaggcct ctgatattac ggacgcgact gccgatggtc atcatagaga caggcgattt 1320 ttctcaatcg gacataatct cctaaatgac cctcaggagt ttgaagcaag attgaattct 1380 ttgatcagaa attttcagaa atttgttaag gctaacggat taatttcctg gctatcgcat 1440 ggtacattgt atggatatct atatgatggt ctgaagtttc cctgggatgt cgaccatgat 1500 ttacagatgc ccattaaaca tttacattac ttgagtcaat atttcaacca atccctaata 1560 ttagaagatc caagagaagg taatggaaga ttcttactag atgtaggaag cgcaattacg 1620 gtaggagttc atgggaacgg cgaaaacaat attgatgctc gtttcatcga tattgactca 1680 ggtatataca ttgacatcac gggacttagc gttagttccg atgcggctaa acagtacatg 1740 tccaaatttg tagaagaaga aagctcgggc gaaagctttt ctgcccttat tgaagactat 1800 aagtttgacg aaaacgacta ttttgacgag gtggatggta gagaaggttt agctaaatat 1860 accatacatg aattaatgga atgggttaat tctcatccag acgactttac ggatgcagaa 1920 aagaatttag tcaccaaaac atacaagaaa gagcttgcaa tttcgagaag cgattatgct 1980 gaaaaagact tgtctccgaa acaaaggtat ttggtaaatg agaagtataa cctttacaat 2040 tgtagaaacc agcatttttc cagtctaaac atcatatcac ccttgagaaa tacaatgttc 2100 agcggtgtgt cagcatttgt tcctaatagg cccatagcaa cattgaataa tgagtataaa 2160 gttccggcaa aatacgggct tttgtcattc caaggtaagg tgtatttacc ggaattcaga 2220 tactggttct cgtttgcaga catgaagaag tttgcaaatt tgcagctgaa agaacccaag 2280 ataacacgac tggaaagtcc cttaaatgat ttaaaattca gcgacataag cctactgata 2340 acaaacattt taaaatgtgg gtttcactcc gtatttgcca gcttatttaa ttcttttgac 2400 agtactgttt acagactcaa agagcttgaa atacagtatg atcctagctt gagtgaggaa 2460 gaaaaaagta gtctattaaa aactctacgg cgaggaatgt caaaaaaaat aaaatcacca 2520 gaaaaagatc cgatcatata tatatacgaa agaaagttat gggaaaacgt ggaaaagttg 2580 ttgaatgcgt caaacatcta caacattgct tcacaagttg agaaggaaaa aggtaaagag 2640 tttgttgaac ggtcccagca agtatatgaa agaaactttg acggcttcag acttcccgat 2700 ggcggcaaca gtaagactgt aaatgatctg aattctaagg gcttaaatct ctttggtgat 2760 aataagaaaa cttcaaacaa tatatttggt tcagaccaaa aatattaa 2808 <210> 3 <211> 1178 <212> PRT <213> Saccharomyces cerevisiae <400> 3 Met Leu Gln Arg Ile Ser Ser Lys Leu His Arg Arg Phe Leu Ser Gly 1 5 10 15 Leu Leu Arg Val Lys His Tyr Pro Leu Arg Arg Ile Leu Leu Pro Leu 20 25 30 Ile Leu Leu Gln Ile Ile Ile Ile Thr Phe Ile Trp Ser Asn Ser Pro 35 40 45 Gln Arg Asn Gly Leu Gly Arg Asp Ala Asp Tyr Leu Leu Pro Asn Tyr 50 55 60 Asn Glu Leu Asp Ser Asp Asp Asp Ser Trp Tyr Ser Ile Leu Thr Ser 65 70 75 80 Ser Phe Lys Asn Asp Arg Lys Ile Gln Phe Ala Lys Thr Leu Tyr Glu 85 90 95 Asn Leu Lys Phe Gly Thr Asn Pro Lys Trp Val Asn Glu Tyr Thr Leu 100 105 110 Gln Asn Asp Leu Leu Ser Val Lys Met Gly Pro Arg Lys Gly Ser Lys 115 120 125 Leu Glu Ser Val Asp Glu Leu Lys Phe Tyr Asp Phe Asp Pro Arg Leu 130 135 140 Thr Trp Ser Val Val Leu Asn His Leu Gln Asn Asn Asp Ala Asp Gln 145 150 155 160 Pro Glu Lys Leu Pro Phe Ser Trp Tyr Asp Trp Thr Thr Phe His Glu 165 170 175 Leu Asn Lys Leu Ile Ser Ile Asp Lys Thr Val Leu Pro Cys Asn Phe 180 185 190 Leu Phe Gln Ser Ala Phe Asp Lys Glu Ser Leu Glu Ala Ile Glu Thr 195 200 205 Glu Leu Gly Glu Pro Leu Phe Leu Tyr Glu Arg Pro Lys Tyr Ala Gln 210 215 220 Lys Leu Trp Tyr Lys Ala Ala Arg Asn Gln Asp Arg Ile Lys Asp Ser 225 230 235 240 Lys Glu Leu Lys Lys His Cys Ser Lys Leu Phe Thr Pro Asp Gly His 245 250 255 Gly Ser Pro Lys Gly Leu Arg Phe Asn Thr Gln Phe Gln Ile Lys Glu 260 265 270 Leu Tyr Asp Lys Val Arg Pro Glu Val Tyr Gln Leu Gln Ala Arg Asn 275 280 285 Tyr Ile Leu Thr Thr Gln Ser His Pro Leu Ser Ile Ser Ile Ile Glu 290 295 300 Ser Asp Asn Ser Thr Tyr Gln Val Pro Leu Gln Thr Glu Lys Ser Lys 305 310 315 320 Asn Leu Val Gln Ser Gly Leu Leu Gln Glu Tyr Ile Asn Asp Asn Ile 325 330 335 Asn Ser Thr Asn Lys Arg Lys Lys Asn Lys Gln Asp Val Glu Phe Asn 340 345 350 His Asn Arg Leu Phe Gln Glu Phe Val Asn Asn Asp Gln Val Asn Ser 355 360 365 Leu Tyr Lys Leu Glu Ile Glu Glu Thr Asp Lys Phe Thr Phe Asp Lys 370 375 380 Asp Leu Val Tyr Leu Ser Pro Ser Asp Phe Lys Phe Asp Ala Ser Lys 385 390 395 400 Lys Ile Glu Glu Leu Glu Glu Gln Lys Lys Leu Tyr Pro Asp Lys Phe 405 410 415 Ser Ala His Asn Glu Asn Tyr Leu Asn Ser Leu Lys Asn Ser Val Lys 420 425 430 Thr Ser Pro Ala Leu Gln Arg Lys Phe Phe Tyr Glu Ala Gly Ala Val 435 440 445 Lys Gln Tyr Lys Gly Met Gly Phe His Arg Asp Lys Arg Phe Phe Asn 450 455 460 Val Asp Thr Leu Ile Asn Asp Lys Gln Glu Tyr Gln Ala Arg Leu Asn 465 470 475 480 Ser Met Ile Arg Thr Phe Gln Lys Phe Thr Lys Ala Asn Gly Ile Ile 485 490 495 Ser Trp Leu Ser His Gly Thr Leu Tyr Gly Tyr Leu Tyr Asn Gly Met 500 505 510 Ala Phe Pro Trp Asp Asn Asp Phe Asp Leu Gln Met Pro Ile Lys His 515 520 525 Leu Gln Leu Leu Ser Gln Tyr Phe Asn Gln Ser Leu Ile Leu Glu Asp 530 535 540 Pro Arg Gln Gly Asn Gly Arg Tyr Phe Leu Asp Val Ser Asp Ser Leu 545 550 555 560 Thr Val Arg Ile Asn Gly Asn Gly Lys Asn Asn Ile Asp Ala Arg Phe 565 570 575 Ile Asp Val Asp Thr Gly Leu Tyr Ile Asp Ile Thr Gly Leu Ala Ser 580 585 590 Thr Ser Ala Pro Ser Arg Asp Tyr Leu Asn Ser Tyr Ile Glu Glu Arg 595 600 605 Leu Gln Glu Glu His Leu Asp Ile Asn Asn Ile Pro Glu Ser Asn Gly 610 615 620 Glu Thr Ala Thr Leu Pro Asp Lys Val Asp Asp Gly Leu Val Asn Met 625 630 635 640 Ala Thr Leu Asn Ile Thr Glu Leu Arg Asp Tyr Ile Thr Ser Asp Glu 645 650 655 Asn Lys Asn His Lys Arg Val Pro Thr Asp Thr Asp Leu Lys Asp Leu 660 665 670 Leu Lys Lys Glu Leu Glu Glu Leu Pro Lys Ser Lys Thr Ile Glu Asn 675 680 685 Lys Leu Asn Pro Lys Gln Arg Tyr Phe Leu Asn Glu Lys Leu Lys Leu 690 695 700 Tyr Asn Cys Arg Asn Asn His Phe Asn Ser Phe Glu Glu Leu Ser Pro 705 710 715 720 Leu Ile Asn Thr Val Phe His Gly Val Pro Ala Leu Ile Pro His Arg 725 730 735 His Thr Tyr Cys Leu His Asn Glu Tyr His Val Pro Asp Arg Tyr Ala 740 745 750 Phe Asp Ala Tyr Lys Asn Thr Ala Tyr Leu Pro Glu Phe Arg Phe Trp 755 760 765 Phe Asp Tyr Asp Gly Leu Lys Lys Cys Ser Asn Ile Asn Ser Trp Tyr 770 775 780 Pro Asn Ile Pro Ser Ile Asn Ser Trp Asn Pro Asn Leu Leu Lys Glu 785 790 795 800 Ile Ser Ser Thr Lys Phe Glu Ser Lys Leu Phe Asp Ser Asn Lys Val 805 810 815 Ser Glu Tyr Ser Phe Lys Asn Leu Ser Met Asp Asp Val Arg Leu Ile 820 825 830 Tyr Lys Asn Ile Pro Lys Ala Gly Phe Ile Glu Val Phe Thr Asn Leu 835 840 845 Tyr Asn Ser Phe Asn Val Thr Ala Tyr Arg Gln Lys Glu Leu Glu Ile 850 855 860 Gln Tyr Cys Gln Asn Leu Thr Phe Ile Glu Lys Lys Lys Leu Leu His 865 870 875 880 Gln Leu Arg Ile Asn Val Ala Pro Lys Leu Ser Ser Pro Ala Lys Asp 885 890 895 Pro Phe Leu Phe Gly Tyr Glu Lys Ala Met Trp Lys Asp Leu Ser Lys 900 905 910 Ser Met Asn Gln Thr Thr Leu Asp Gln Val Thr Lys Ile Val His Glu 915 920 925 Glu Tyr Val Gly Lys Ile Ile Asp Leu Ser Glu Ser Leu Lys Tyr Arg 930 935 940 Asn Phe Ser Leu Phe Asn Ile Thr Phe Asp Glu Thr Gly Thr Thr Leu 945 950 955 960 Asp Asp Asn Thr Glu Asp Tyr Thr Pro Ala Asn Thr Val Glu Val Asn 965 970 975 Pro Val Asp Phe Lys Ser Asn Leu Asn Phe Ser Ser Asn Ser Phe Leu 980 985 990 Asp Leu Asn Ser Tyr Gly Leu Asp Leu Phe Ala Pro Thr Leu Ser Asp 995 1000 1005 Val Asn Arg Lys Gly Ile Gln Met Phe Asp Lys Asp Pro Ile Ile Val 1010 1015 1020 Tyr Glu Asp Tyr Ala Tyr Ala Lys Leu Leu Glu Glu Arg Lys Arg Arg 1025 1030 1035 1040 Glu Lys Lys Lys Lys Glu Glu Glu Glu Lys Lys Lys Lys Glu Glu Glu 1045 1050 1055 Glu Lys Lys Lys Lys Glu Glu Glu Glu Lys Lys Lys Lys Glu Glu Glu 1060 1065 1070 Glu Lys Lys Lys Lys Glu Glu Glu Glu Lys Lys Lys Lys Glu Glu Glu 1075 1080 1085 Glu Lys Lys Lys Gln Glu Glu Glu Glu Lys Lys Lys Lys Glu Glu Glu 1090 1095 1100 Glu Lys Lys Lys Gln Glu Glu Gly Glu Lys Met Lys Asn Glu Asp Glu 1105 1110 1115 1120 Glu Asn Lys Lys Asn Glu Asp Glu Glu Lys Lys Lys Asn Glu Glu Glu 1125 1130 1135 Glu Lys Lys Lys Gln Glu Glu Lys Asn Lys Lys Asn Glu Asp Glu Glu 1140 1145 1150 Lys Lys Lys Gln Glu Glu Glu Glu Lys Lys Lys Asn Glu Glu Glu Glu 1155 1160 1165 Lys Lys Lys Gln Glu Glu Gly His Ser Asn 1170 1175 <210> 4 <211> 3534 <212> DNA <213> Saccharomyces cerevisiae <400> 4 atgcttcagc gaatatcatc taaacttcac aggcggttct tatctggcct gctgcgtgtc 60 aagcactacc cattaaggcg cattctcctt ccactgattc tactgcagat catcattata 120 acgtttatct ggtcaaattc accgcagcgt aacggacttg ggcgggacgc tgattacctt 180 ctaccaaatt acaacgaact tgacagtgat gatgattcct ggtatagcat cctgacttcg 240 tctttcaaaa acgatcgcaa gatccagttc gctaagacat tatacgaaaa tttaaaattc 300 ggcaccaacc ctaaatgggt caatgaatat actctgcaaa atgacctgct ctcggtcaaa 360 atgggccctc gaaagggcag taagctcgaa tccgtggatg agttgaagtt ttacgacttc 420 gaccctcgtc tcacgtggtc cgttgtgctg aaccatttgc aaaataatga cgcagatcag 480 ccagaaaagt tacccttttc atggtacgac tggacaacct tccacgagct gaataagctg 540 atttccatag ataaaactgt tctgccctgc aattttcttt tccagtccgc tttcgacaaa 600 gagtctttag aggccattga gacagagctc ggcgaacctt tgttcctata cgaaagacca 660 aagtacgcgc agaaactgtg gtacaaggcc gctagaaacc aggacagaat caaagactca 720 aaggaactaa aaaagcattg ttccaagcta ttcactccag acgggcatgg ctctcctaag 780 ggtttaagat ttaatacgca atttcaaata aaggagctgt atgataaagt tagacccgaa 840 gtttaccaat tgcaggcaag aaactacatt ttgactacac agtcgcatcc actatccatt 900 tccatcatcg aatcagataa ttccacgtat caagtcccct tgcaaactga aaaatcaaaa 960 aacttggtgc aatccggcct gttgcaggaa tatattaatg ataacattaa ttctacgaac 1020 aagagaaaga aaaataaaca ggacgtagaa ttcaaccata acaggctttt ccaggaattc 1080 gtcaataacg accaagttaa ctccctatac aaactggaaa ttgaagaaac tgataaattc 1140 acttttgata aagatttggt ttatttatcc ccttcggatt tcaagttcga tgcctccaaa 1200 aaaattgaag agttagagga acagaagaaa ctctatccgg acaaattttc cgctcataat 1260 gagaattatc tgaacagttt gaagaattcc gtaaagacaa gccctgcatt gcaaagaaag 1320 ttcttctatg aggctggtgc cgtgaagcaa tataaaggta tggggttcca tcgtgacaag 1380 aggttcttca atgttgatac attaatcaat gataaacaag aataccaggc tagattgaac 1440 tcaatgatca gaacattcca aaagtttact aaagccaacg gcatcatatc ttggttgtct 1500 cacggaacgc tgtacggcta tctttacaat ggaatggctt tcccttggga taacgatttc 1560 gacttgcaaa tgcccattaa gcatttacaa ttgctcagtc aatacttcaa ccaatctctt 1620 atattggaag acccaagaca gggtaatgga cgttatttcc tagacgtcag cgactccttg 1680 acagtaagaa ttaacggtaa cggtaaaaac aatatcgatg caagattcat tgacgtcgac 1740 accggccttt acattgatat taccggtcta gctagcactt ctgcccctag tagggattac 1800 ttgaattctt atattgaaga gcggttgcaa gaggaacatt tggatatcaa taatatccct 1860 gaatcgaacg gtgagaccgc tactttgccc gacaaagtag atgatgggtt agtcaatatg 1920 gctacactaa acatcactga gctacgtgat tacattacca gcgacgaaaa taaaaatcat 1980 aaaagagtcc ccactgatac tgatttgaaa gatcttttga aaaaggaact ggaagagtta 2040 ccaaagtcta agaccattga aaacaagttg aatcctaaac aaagatattt tctcaacgaa 2100 aaacttaaac tttacaattg tagaaacaac cattttaact cgttcgagga actatctccc 2160 ttaatcaata ctgttttcca tggtgtgcca gcgttgattc ctcacagaca tacctactgc 2220 ttgcacaatg aatatcatgt acctgataga tatgcatttg atgcttacaa aaatactgct 2280 tatttgcccg aatttagatt ttggttcgac tatgacgggt taaagaaatg cagtaatatt 2340 aattcatggt atccaaacat ccccagtatt aattcatgga atccgaacct cttgaaagaa 2400 atatcgtcta cgaaatttga gtcgaaactt tttgattcca acaaagtctc tgaatactct 2460 ttcaaaaacc tatccatgga tgatgttcgc ttaatttata aaaatattcc aaaagctggc 2520 tttatcgagg tatttactaa cttgtacaat tccttcaatg tcactgcata taggcaaaag 2580 gaattggaaa ttcaatactg ccaaaacctg acatttattg aaaaaaagaa attattacat 2640 caattgcgca ttaatgttgc tcctaagtta agctcccctg caaaggaccc atttcttttt 2700 ggttatgaaa aagctatgtg gaaggattta tcaaaatcta tgaaccagac tacattagat 2760 caagttacca agattgttca tgaagaatat gtcggaaaaa ttattgatct gtccgaaagt 2820 ttgaaataca ggaatttttc acttttcaac attacttttg atgaaactgg aacaactcta 2880 gatgataaca cagaagatta tactcctgct aatactgttg aagtaaatcc tgtggatttt 2940 aaatcaaatt taaactttag tagcaactcc tttttggatt taaattcata tggtttagac 3000 ctttttgcgc caactttatc cgacgttaac agaaagggta ttcaaatgtt tgataaggac 3060 cctattattg tatacgagga ctatgcttat gccaagttac ttgaagaaag aaagcggagg 3120 gagaagaaga agaaggagga agaggagaag aagaagaagg aagaagagga aaagaagaag 3180 aaggaagaag aagaaaagaa aaagaaggaa gaggaagaga agaaaaagaa ggaagaagaa 3240 gagaagaaaa agaaggaaga agaagaaaag aagaagcagg aggaagagga gaaaaagaag 3300 aaggaagaag aagagaagaa gaagcaggaa gaaggagaaa agatgaagaa tgaagatgaa 3360 gaaaataaga agaatgaaga tgaagaaaag aagaagaacg aagaagagga aaaaaagaag 3420 caggaagaga aaaacaagaa gaatgaagat gaagaaaaga agaagcagga agaggaagaa 3480 aagaagaaga acgaagaaga ggaaaaaaag aagcaggagg aggggcacag caat 3534 <210> 5 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Och1_pUG72_F <400> 5 atgtctagga agttgtccca cctgatcgct acaaggaaat caaaatacgc tgcaggtcga 60 caacc 65 <210> 6 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Och1_pUG72_R <400> 6 ttatttatga cctgcatttt tatcagcatc ttctttccag ctcccactag tggatctgat 60 atcacc 66 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Och1_CF <400> 7 aatggggagc gctgattctc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Och1_CR <400> 8 tctacggaag gacgttgaga 20 <210> 9 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Mnn1_pUG6_F <400> 9 aacgtaatct tgcggtattt aacgctagtt taagaaagtg ttactgtgta tacgctgcag 60 gtcgacaacc 70 <210> 10 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Mnn1_pUG6_R <400> 10 gttcacaaag gctagtacca taaacagtta gaaaaaacac tggttaatgc actagtggat 60 ctgatatcac c 71 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mnn1_CF <400> 11 atcattgcga ggtctcaatt gg 22 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Mnn1_CR <400> 12 gattagaaaa actcatcgag catcaaatg 29 <210> 13 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_F <400> 13 acaacgtcac tattccttca cacaaataaa ctaattagtt atgcttcagc gtacgctgca 60 ggtcgacaac c 71 <210> 14 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_R <400> 14 aggaaaggct atagaaatga agagattcat gaattttcag tcaggttcta ctagtggatc 60 tgatatcacc 70 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Leu2_CF <400> 15 agccttgtca agagaccaga 20 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_CR <400> 16 attgtttgcc acttatcact ggcg 24 <210> 17 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Mnn6_F <400> 17 agcgttcacc caaccttttg tgccctttag tgaagataag ataaggtaag tacgctgcag 60 gtcgacaacc 70 <210> 18 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Mnn6_R <400> 18 tatatattca tatgtagaag attattgttc ttatacatca gtgttttgat actagtggat 60 ctgatatcac c 71 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mnn6_CF <400> 19 gctctcgtga gacacgagtt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ura3_CR <400> 20 cccgtcaatt agttgcacca 20 <210> 21 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Mnn14_F <400> 21 atgatgttat cactgcgcag gttctccatg tacgttttga gatctctgcg tacgctgcag 60 gtcgacaacc 70 <210> 22 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Mnn14_R <400> 22 ttaatatttt tggtctgaac caaatatatt gtttgaagtt ttcttattat actagtggat 60 ctgatatcac c 71 <210> 23 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Yur1_F <400> 23 atggcaaaag gaggctcgct atacatcgtt ggcatattct taccaatatg tacgctgcag 60 gtcgacaacc 70 <210> 24 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Yur1_R <400> 24 ttaaatctcg tcttgctctt cttttaagaa atatttgccg ctaccgtttt actagtggat 60 ctgatatcac c 71 <210> 25 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Ktr2_F <400> 25 atgcaaatct gcaaggtatt tcttacacag gttaaaaaac tactttttgt tacgctgcag 60 gtcgacaacc 70 <210> 26 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Ktr2_R <400> 26 ctatgaatcg tgtttgagga agtatttacc gctgccgtcc ttccaccatc actagtggat 60 ctgatatcac c 71 <210> 27 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Ktr4_F <400> 27 atgaggtttc tttcaaaaag gatactgaaa cctgtacttt cagtgatcat tacgctgcag 60 gtcgacaacc 70 <210> 28 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Ktr4_R <400> 28 tcaatacatt tctaactctt cctcagacat agagtgtctt atccaggttg actagtggat 60 ctgatatcac c 71 <210> 29 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Ktr5_F <400> 29 atgttgctaa taagaaggac gataaatgca tttctgggat gtatccattg tacgctgcag 60 gtcgacaacc 70 <210> 30 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Ktr5_R <400> 30 ctagtttccg aactgtctta gatagtcttc ccttatgtgc tcctccattt actagtggat 60 ctgatatcac c 71 <210> 31 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Ktr7_F <400> 31 atggctataa gattgaatcc aaaagtcaga aggttcttgc tggataagtg tacgctgcag 60 gtcgacaacc 70 <210> 32 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Ktr7_R <400> 32 ctattcaatt actctaaaat tttctcttct gatctcttca atcacgtctt actagtggat 60 ctgatatcac c 71 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Mnn14_CF <400> 33 cgaagatcaa gtaagagtgc acttg 25 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Yur1_CF <400> 34 atctgtcact gcttattcat atcatc 26 <210> 35 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Ktr2_CF <400> 35 atctcttcag gtatgtgaca cctata 26 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Ktr4_CF <400> 36 caacggaacg agctctataa gacg 24 <210> 37 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Ktr5_CF <400> 37 acactttaag catgcggtgt gtgga 25 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Ktr7_CF <400> 38 gtatacatca ggctaacaat ctgtga 26 <210> 39 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> GAPDHp-F1 <400> 39 agtcgaattc atactagcgt tgaatgttag cg 32 <210> 40 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> GAPDHp-R1 <400> 40 agtcggtacc tttgtttgtt tatgtgtgtt tattc 35 <210> 41 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> GAPDHt-F1 <400> 41 agtcggtacc ggatcctcta gagtgaattt actttaaatc ttgcat 46 <210> 42 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> GAPDHt-R1 <400> 42 agtcctgcag atccacaatg tatcaggtat ct 32 <210> 43 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_F <400> 43 cgcggatcca tgcttcagcg aatatcatct aaac 34 <210> 44 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_R <400> 44 ggactagttt aattgctgtg cccctcctc 29 <210> 45 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Y-Mnn14-F <400> 45 aacacacata aacaaacaaa ggtaccatga tgttatcact gcgcagg 47 <210> 46 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Y-Mnn14-R <400> 46 aaattcactc tagaggatcc ggtaccttaa tatttttggt ctgaaccaaa 50 <210> 47 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> p-Gas1-F <400> 47 tacacgtact tagtcgctga agctcttcta tgatgttgtt taaatccctt tcaaag 56 <210> 48 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> p-Gas1-R-1 <400> 48 gtgatgatga tgatggtggt ggtgagaacc ccctccacca ctggattcag ttccggaacc 60 60 <210> 49 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> p-Gas1-R-2 <400> 49 aggtacgaac tcgattgacg gctcttctac cgtgatgatg atgatggtgg tg 52 <110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> An essential gene MNN14 for mannosylphosphorylation in Saccharomyces cerevisiae and a method for producing recombinant glycoprotein using a MNN14-defected microorganism <130> KPA150188-KR-P1-D1 <150> 10-2015-0064855 <151> 2015-05-08 <160> 49 <170> KopatentIn 2.0 <210> 1 <211> 935 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Met Met Leu Ser Leu Arg Arg Phe Ser Met Tyr Val Leu Arg Ser Leu 1 5 10 15 Arg Leu His Phe Lys Lys Ile Ile Ile Thr Leu Leu Thr Ile Gln Leu 20 25 30 Leu Phe Ile Thr Ile Phe Val Leu Gly Gly Arg Ser Ser Ile Ile Asp 35 40 45 Gly Asn Trp Lys Ser Phe Met Ala Leu Phe Phe Lys Pro Leu Ala Tyr 50 55 60 Thr Asn Arg Asn Asn Asn His Ala Ser Phe Asp Leu Arg Ser Lys Asp 65 70 75 80 Asn Val Ala Lys Leu Tyr Glu Lys Met Asn Phe Asp Thr Ser Gly Lys 85 90 95 Trp Ile Asp Thr Tyr Thr Leu Lys Asn Asn Leu Leu Thr Val Lys Met 100 105 110 Gly Pro Glu Lys Gly Gln Val Leu Asp Ser Val Asp Glu Leu Arg Tyr 115 120 125 Tyr Asp Asn Asp Pro Arg Leu Val Trp Ser Val Leu Leu Asp His Leu 130 135 140 Leu Glu Ser Asp Ser Asn Glu Tyr Ala Phe Ser Trp Tyr Asp Trp Ala 145 150 155 160 Asn Phe Asp Ser Thr Asn Lys Leu Ile Ala Leu Arg His Thr Asn Ile 165 170 175 Ser Cys Gln Phe Val Cys Glu Gly Ala Phe Asp Lys Asn Val Leu Glu 180 185 190 Met Val Glu Ser Glu Val Gln Glu Pro Leu Phe Val Thr Asn Arg Asn 195 200 205 Lys Tyr Asp Glu Ser Leu Trp Tyr Asn Arg Val Arg Lys Val Val Asp 210 215 220 Ser Asn Ser Val Gln Gln Ala Ile His Asp His Cys Met Asn Asn Asp 225 230 235 240 Ala Tyr Ser Asn Gly Thr Pro Phe Glu Leu Pro Phe Ile Ile Ser Glu 245 250 255 Ile Ser Glu Arg Leu Arg Pro Glu Val Tyr Asp Leu Gln Ala Lys Asn 260 265 270 His Leu Leu Tyr Ser Asn Phe Thr Pro Leu Ser Leu Thr Val Leu Asp 275 280 285 Ser Asp Lys Asp Ala Tyr Arg Ile Asn Leu Lys Thr Thr Asp Ser Ser 290 295 300 Lys Ser Asn Ile Val Gln Thr Asn Leu Leu Gln Asn Tyr Ile Lys Arg 305 310 315 320 His Arg Asn Glu Met Val Asn Gly Asp Leu Ile Phe Asn His Thr Ser 325 330 335 Met Phe Glu Lys Phe Leu His His Gly Ser Thr Lys Lys Arg Lys Leu 340 345 350 Asp Val Glu Ala Leu Asp Lys Thr Ile Tyr Ala Gly Glu Tyr Leu Glu 355 360 365 Leu Ser Pro Ser Asp Phe Gln Phe Asn Ala Lys Glu Arg Ile Ile Glu 370 375 380 Leu Glu Thr Arg Leu Arg Ser Glu Gly Leu Pro Ser His Asp Thr His 385 390 395 400 Tyr Leu Arg Ser Leu Lys Thr Ser Val Asn Thr Ser Pro Ala Leu Gln 405 410 415 Gln Lys Tyr Phe Ala Glu Ala Ser Asp Ile Thr Asp Ala Thr Ala Asp 420 425 430 Gly His His Arg Asp Arg Arg Phe Phe Ser Ile Gly His Asn Leu Leu 435 440 445 Asn Asp Pro Gln Glu Phe Glu Ala Arg Leu Asn Ser Leu Ile Arg Asn 450 455 460 Phe Gln Lys Phe Val Lys Ala Asn Gly Leu Ile Ser Trp Leu Ser His 465 470 475 480 Gly Thr Leu Tyr Gly Tyr Leu Tyr Asp Gly Leu Lys Phe Pro Trp Asp 485 490 495 Val Asp His Asp Leu Gln Met Pro Ile Lys His Leu His Tyr Leu Ser 500 505 510 Gln Tyr Phe Asn Gln Ser Leu Ile Leu Glu Asp Pro Arg Glu Gly Asn 515 520 525 Gly Arg Phe Leu Leu Asp Val Gly Ser Ala Ile Thr Val Gly Val His 530 535 540 Gly Asn Gly Glu Asn Asn Ile Asp Ala Arg Phe Ile Asp Ile Asp Ser 545 550 555 560 Gly Ile Tyr Ile Asp Ile Thr Gly Leu Ser Val Ser Ser Asp Ala Ala 565 570 575 Lys Gln Tyr Met Ser Lys Phe Val Glu Glu Glu Ser Ser Gly Glu Ser 580 585 590 Phe Ser Ala Leu Ile Glu Asp Tyr Lys Phe Asp Glu Asn Asp Tyr Phe 595 600 605 Asp Glu Val Asp Gly Arg Glu Gly Leu Ala Lys Tyr Thr Ile His Glu 610 615 620 Leu Met Glu Trp Val Asn Ser His Pro Asp Asp Phe Thr Asp Ala Glu 625 630 635 640 Lys Asn Leu Val Thr Lys Thr Tyr Lys Lys Glu Leu Ala Ile Ser Arg 645 650 655 Ser Asp Tyr Ala Glu Lys Asp Leu Ser Pro Lys Gln Arg Tyr Leu Val 660 665 670 Asn Glu Lys Tyr Asn Leu Tyr Asn Cys Arg Asn Gln His Phe Ser Ser 675 680 685 Leu Asn Ile Ile Ser Pro Leu Arg Asn Thr Met Phe Ser Gly Val Ser 690 695 700 Ala Phe Val Pro Asn Arg Pro Ile Ala Thr Leu Asn Asn Glu Tyr Lys 705 710 715 720 Val Pro Ala Lys Tyr Gly Leu Leu Ser Phe Gln Gly Lys Val Tyr Leu 725 730 735 Pro Glu Phe Arg Tyr Trp Phe Ser Phe Ala Asp Met Lys Lys Phe Ala 740 745 750 Asn Leu Gln Leu Lys Glu Pro Lys Ile Thr Arg Leu Glu Ser Pro Leu 755 760 765 Asn Asp Leu Lys Phe Ser Asp Ile Ser Leu Leu Ile Thr Asn Ile Leu 770 775 780 Lys Cys Gly Phe His Ser Val Phe Ala Ser Leu Phe Asn Ser Phe Asp 785 790 795 800 Ser Thr Val Tyr Arg Leu Lys Glu Leu Glu Ile Gln Tyr Asp Pro Ser 805 810 815 Leu Ser Glu Glu Glu Lys Ser Ser Leu Leu Lys Thr Leu Arg Arg Gly 820 825 830 Met Ser Lys Lys Ile Lys Ser Pro Glu Lys Asp Pro Ile Ile Tyr Ile 835 840 845 Tyr Glu Arg Lys Leu Trp Glu Asn Val Glu Lys Leu Leu Asn Ala Ser 850 855 860 Asn Ile Tyr Asn Ile Ala Ser Gln Val Glu Lys Glu Lys Gly Lys Glu 865 870 875 880 Phe Val Glu Arg Ser Gln Gln Val Tyr Glu Arg Asn Phe Asp Gly Phe 885 890 895 Arg Leu Pro Asp Gly Gly Asn Ser Lys Thr Val Asn Asp Leu Asn Ser 900 905 910 Lys Gly Leu Asn Leu Phe Gly Asp Asn Lys Lys Thr Ser Asn Asn Ile 915 920 925 Phe Gly Ser Asp Gln Lys Tyr 930 935 <210> 2 <211> 2808 <212> DNA <213> Saccharomyces cerevisiae <400> 2 atgatgttat cactgcgcag gttctccatg tacgttttga gatctctgcg gcttcacttt 60 aaaaagataa tcattactct tctaactatc cagttactat tcattaccat atttgtattg 120 ggcggtcgct cgtcgattat tgacggtaac tggaagtcat tcatggcgct ctttttcaaa 180 ccgcttgctt acactaacag aaacaacaac catgcttctt tcgatctgag atcaaaagac 240 aacgtagcca aactttacga aaaaatgaat tttgatactt caggcaaatg gatcgacacg 300 tacaccttga agaataatct tctcactgtg aaaatgggtc ctgaaaaagg gcaagttctt 360 gattcggtag atgaattgag atattacgat aacgacccaa ggctggtatg gtcagtttta 420 ctagatcact tattagaatc agattccaat gaatacgcat tttcgtggta cgattgggct 480 aattttgact ctacaaacaa actcattgca ctgagacaca cgaacatatc ttgccagttc 540 gtttgcgagg gtgcctttga taaaaatgtg ctagaaatgg tagagagtga agtccaagag 600 cctttattcg tcacaaatag gaataaatat gacgaatcgc tctggtacaa cagggtaaga 660 aaggttgtcg attctaattc tgtgcagcaa gccatacatg atcactgcat gaataatgac 720 gcgtattcca atggtactcc cttcgaattg ccttttatca taagcgaaat ttctgaaagg 780 ttgaggccag aagtgtatga cttacaagcc aaaaaccact tgttatattc taactttact 840 ccactgtcat taaccgtact ggacagcgat aaagatgcat acagaatcaa tttgaagaca 900 acagactctt ccaaatcaaa tatagtacag acaaatctac tacagaatta cattaagagg 960 cacagaaatg aaatggtaaa tggcgacctc attttcaacc acacttccat gtttgaaaaa 1020 tttttacatc atggatccac taaaaaaagg aaacttgacg ttgaagcgtt ggataaaaca 1080 atatacgctg gagagtatct agaactatca ccatctgatt tccaattcaa tgcaaaagag 1140 aggatcattg aattagagac caggctcagg tctgaaggcc taccatctca tgatacccac 1200 tatttacgaa gtttaaagac gtccgtaaat acgtcccctg cattacagca aaagtatttc 1260 gcagaggcct ctgatattac ggacgcgact gccgatggtc atcatagaga caggcgattt 1320 ttctcaatcg gacataatct cctaaatgac cctcaggagt ttgaagcaag attgaattct 1380 ttgatcagaa attttcagaa atttgttaag gctaacggat taatttcctg gctatcgcat 1440 ggtacattgt atggatatct atatgatggt ctgaagtttc cctgggatgt cgaccatgat 1500 ttacagatgc ccattaaaca tttacattac ttgagtcaat atttcaacca atccctaata 1560 ttagaagatc caagagaagg taatggaaga ttcttactag atgtaggaag cgcaattacg 1620 gtaggagttc atgggaacgg cgaaaacaat attgatgctc gtttcatcga tattgactca 1680 ggtatataca ttgacatcac gggacttagc gttagttccg atgcggctaa acagtacatg 1740 tccaaatttg tagaagaaga aagctcgggc gaaagctttt ctgcccttat tgaagactat 1800 aagtttgacg aaaacgacta ttttgacgag gtggatggta gagaaggttt agctaaatat 1860 accatacatg aattaatgga atgggttaat tctcatccag acgactttac ggatgcagaa 1920 aagaatttag tcaccaaaac atacaagaaa gagcttgcaa tttcgagaag cgattatgct 1980 gaaaaagact tgtctccgaa acaaaggtat ttggtaaatg agaagtataa cctttacaat 2040 tgtagaaacc agcatttttc cagtctaaac atcatatcac ccttgagaaa tacaatgttc 2100 agcggtgtgt cagcatttgt tcctaatagg cccatagcaa cattgaataa tgagtataaa 2160 gttccggcaa aatacgggct tttgtcattc caaggtaagg tgtatttacc ggaattcaga 2220 tactggttct cgtttgcaga catgaagaag tttgcaaatt tgcagctgaa agaacccaag 2280 ataacacgac tggaaagtcc cttaaatgat ttaaaattca gcgacataag cctactgata 2340 acaaacattt taaaatgtgg gtttcactcc gtatttgcca gcttatttaa ttcttttgac 2400 agtactgttt acagactcaa agagcttgaa atacagtatg atcctagctt gagtgaggaa 2460 gaaaaaagta gtctattaaa aactctacgg cgaggaatgt caaaaaaaat aaaatcacca 2520 gaaaaagatc cgatcatata tatatacgaa agaaagttat gggaaaacgt ggaaaagttg 2580 ttgaatgcgt caaacatcta caacattgct tcacaagttg agaaggaaaa aggtaaagag 2640 tttgttgaac ggtcccagca agtatatgaa agaaactttg acggcttcag acttcccgat 2700 ggcggcaaca gtaagactgt aaatgatctg aattctaagg gcttaaatct ctttggtgat 2760 aataagaaaa cttcaaacaa tatatttggt tcagaccaaa aatattaa 2808 <210> 3 <211> 1178 <212> PRT <213> Saccharomyces cerevisiae <400> 3 Met Leu Gln Arg Ile Ser Ser Lys Leu His Arg Arg Phe Leu Ser Gly 1 5 10 15 Leu Leu Arg Val Lys His Tyr Pro Leu Arg Arg Ile Leu Leu Pro Leu 20 25 30 Ile Leu Leu Gln Ile Ile Ile Ile Thr Phe Ile Trp Ser Asn Ser Pro 35 40 45 Gln Arg Asn Gly Leu Gly Arg Asp Ala Asp Tyr Leu Leu Pro Asn Tyr 50 55 60 Asn Glu Leu Asp Ser Asp Asp Asp Ser Trp Tyr Ser Ile Leu Thr Ser 65 70 75 80 Ser Phe Lys Asn Asp Arg Lys Ile Gln Phe Ala Lys Thr Leu Tyr Glu 85 90 95 Asn Leu Lys Phe Gly Thr Asn Pro Lys Trp Val Asn Glu Tyr Thr Leu 100 105 110 Gln Asn Asp Leu Leu Ser Val Lys Met Gly Pro Arg Lys Gly Ser Lys 115 120 125 Leu Glu Ser Val Asp Glu Leu Lys Phe Tyr Asp Phe Asp Pro Arg Leu 130 135 140 Thr Trp Ser Val Val Leu Asn His Leu Gln Asn Asn Asp Ala Asp Gln 145 150 155 160 Pro Glu Lys Leu Pro Phe Ser Trp Tyr Asp Trp Thr Thr Phe His Glu 165 170 175 Leu Asn Lys Leu Ile Ser Ile Asp Lys Thr Val Leu Pro Cys Asn Phe 180 185 190 Leu Phe Gln Ser Ala Phe Asp Lys Glu Ser Leu Glu Ala Ile Glu Thr 195 200 205 Glu Leu Gly Glu Pro Leu Phe Leu Tyr Glu Arg Pro Lys Tyr Ala Gln 210 215 220 Lys Leu Trp Tyr Lys Ala Ala Arg Asn Gln Asp Arg Ile Lys Asp Ser 225 230 235 240 Lys Glu Leu Lys Lys His Cys Ser Lys Leu Phe Thr Pro Asp Gly His 245 250 255 Gly Ser Pro Lys Gly Leu Arg Phe Asn Thr Gln Phe Gln Ile Lys Glu 260 265 270 Leu Tyr Asp Lys Val Arg Pro Glu Val Tyr Gln Leu Gln Ala Arg Asn 275 280 285 Tyr Ile Leu Thr Thr Gln Ser His Pro Leu Ser Ile Ser Ile Ile Glu 290 295 300 Ser Asp Asn Ser Thr Tyr Gln Val Pro Leu Gln Thr Glu Lys Ser Lys 305 310 315 320 Asn Leu Val Gln Ser Gly Leu Leu Gln Glu Tyr Ile Asn Asp Asn Ile 325 330 335 Asn Ser Thr Asn Lys Arg Lys Lys Asn Lys Gln Asp Val Glu Phe Asn 340 345 350 His Asn Arg Leu Phe Gln Glu Phe Val Asn Asn Asp Gln Val Asn Ser 355 360 365 Leu Tyr Lys Leu Glu Ile Glu Glu Thr Asp Lys Phe Thr Phe Asp Lys 370 375 380 Asp Leu Val Tyr Leu Ser Pro Ser Asp Phe Lys Phe Asp Ala Ser Lys 385 390 395 400 Lys Ile Glu Glu Leu Glu Glu Gln Lys Lys Leu Tyr Pro Asp Lys Phe 405 410 415 Ser Ala His Asn Glu Asn Tyr Leu Asn Ser Leu Lys Asn Ser Val Lys 420 425 430 Thr Ser Pro Ala Leu Gln Arg Lys Phe Phe Tyr Glu Ala Gly Ala Val 435 440 445 Lys Gln Tyr Lys Gly Met Gly Phe His Arg Asp Lys Arg Phe Phe Asn 450 455 460 Val Asp Thr Leu Ile Asn Asp Lys Gln Glu Tyr Gln Ala Arg Leu Asn 465 470 475 480 Ser Met Ile Arg Thr Phe Gln Lys Phe Thr Lys Ala Asn Gly Ile Ile 485 490 495 Ser Trp Leu Ser His Gly Thr Leu Tyr Gly Tyr Leu Tyr Asn Gly Met 500 505 510 Ala Phe Pro Trp Asp Asn Asp Phe Asp Leu Gln Met Pro Ile Lys His 515 520 525 Leu Gln Leu Leu Ser Gln Tyr Phe Asn Gln Ser Leu Ile Leu Glu Asp 530 535 540 Pro Arg Gln Gly Asn Gly Arg Tyr Phe Leu Asp Val Ser Asp Ser Leu 545 550 555 560 Thr Val Arg Ile Asn Gly Asn Gly Lys Asn Asn Ile Asp Ala Arg Phe 565 570 575 Ile Asp Val Asp Thr Gly Leu Tyr Ile Asp Ile Thr Gly Leu Ala Ser 580 585 590 Thr Ser Ala Pro Ser Arg Asp Tyr Leu Asn Ser Tyr Ile Glu Glu Arg 595 600 605 Leu Gln Glu Glu His Leu Asp Ile Asn Asn Ile Pro Glu Ser Asn Gly 610 615 620 Glu Thr Ala Thr Leu Pro Asp Lys Val Asp Asp Gly Leu Val Asn Met 625 630 635 640 Ala Thr Leu Asn Ile Thr Glu Leu Arg Asp Tyr Ile Thr Ser Asp Glu 645 650 655 Asn Lys Asn His Lys Arg Val Pro Thr Asp Thr Asp Leu Lys Asp Leu 660 665 670 Leu Lys Lys Glu Leu Glu Glu Leu Pro Lys Ser Lys Thr Ile Glu Asn 675 680 685 Lys Leu Asn Pro Lys Gln Arg Tyr Phe Leu Asn Glu Lys Leu Lys Leu 690 695 700 Tyr Asn Cys Arg Asn Asn His Phe Asn Ser Phe Glu Glu Leu Ser Pro 705 710 715 720 Leu Ile Asn Thr Val Phe His Gly Val Pro Ala Leu Ile Pro His Arg 725 730 735 His Thr Tyr Cys Leu His Asn Glu Tyr His Val Pro Asp Arg Tyr Ala 740 745 750 Phe Asp Ala Tyr Lys Asn Thr Ala Tyr Leu Pro Glu Phe Arg Phe Trp 755 760 765 Phe Asp Tyr Asp Gly Leu Lys Lys Cys Ser Asn Ile Asn Ser Trp Tyr 770 775 780 Pro Asn Ile Pro Ser Ile Asn Ser Trp Asn Pro Asn Leu Leu Lys Glu 785 790 795 800 Ile Ser Ser Thr Lys Phe Glu Ser Lys Leu Phe Asp Ser Asn Lys Val 805 810 815 Ser Glu Tyr Ser Phe Lys Asn Leu Ser Met Asp Asp Val Arg Leu Ile 820 825 830 Tyr Lys Asn Ile Pro Lys Ala Gly Phe Ile Glu Val Phe Thr Asn Leu 835 840 845 Tyr Asn Ser Phe Asn Val Thr Ala Tyr Arg Gln Lys Glu Leu Glu Ile 850 855 860 Gln Tyr Cys Gln Asn Leu Thr Phe Ile Glu Lys Lys Lys Leu Leu His 865 870 875 880 Gln Leu Arg Ile Asn Val Ala Pro Lys Leu Ser Ser Pro Ala Lys Asp 885 890 895 Pro Phe Leu Phe Gly Tyr Glu Lys Ala Met Trp Lys Asp Leu Ser Lys 900 905 910 Ser Met Asn Gln Thr Thr Leu Asp Gln Val Thr Lys Ile Val His Glu 915 920 925 Glu Tyr Val Gly Lys Ile Ile Asp Leu Ser Glu Ser Leu Lys Tyr Arg 930 935 940 Asn Phe Ser Leu Phe Asn Ile Thr Phe Asp Glu Thr Gly Thr Thr Leu 945 950 955 960 Asp Asp Asn Thr Glu Asp Tyr Thr Pro Ala Asn Thr Val Glu Val Asn 965 970 975 Pro Val Asp Phe Lys Ser Asn Leu Asn Phe Ser Ser Asn Ser Phe Leu 980 985 990 Asp Leu Asn Ser Tyr Gly Leu Asp Leu Phe Ala Pro Thr Leu Ser Asp 995 1000 1005 Val Asn Arg Lys Gly Ile Gln Met Phe Asp Lys Asp Pro Ile Ile Val 1010 1015 1020 Tyr Glu Asp Tyr Ala Tyr Ala Lys Leu Leu Glu Glu Arg Lys Arg Arg 1025 1030 1035 1040 Glu Lys Lys Lys Lys Glu Glu Glu Glu Glu Lys Lys Lys Lys Glu Glu Glu 1045 1050 1055 Glu Lys Lys Lys Lys Glu Glu Glu Glu Glu Lys Lys Lys Lys Glu Glu Glu 1060 1065 1070 Glu Lys Lys Lys Lys Glu Glu Glu Glu Glu Lys Lys Lys Lys Glu Glu Glu 1075 1080 1085 Glu Lys Lys Lys Gln Glu Glu Glu Glu Glu Lys Lys Lys Lys Glu Glu Glu 1090 1095 1100 Glu Lys Lys Lys Gln Glu Glu Gly Glu Lys Met Lys Asn Glu Asp Glu 1105 1110 1115 1120 Glu Asn Lys Lys Asn Glu Asp Glu Glu Lys Lys Lys Asn Glu Glu Glu 1125 1130 1135 Glu Lys Lys Lys Gln Glu Glu Lys Asn Lys Lys Asn Glu Asp Glu Glu 1140 1145 1150 Lys Lys Lys Gln Glu Glu Glu Glu Lys Lys Lys Asn Glu Glu Glu Glu 1155 1160 1165 Lys Lys Lys Gln Glu Glu Gly His Ser Asn 1170 1175 <210> 4 <211> 3534 <212> DNA <213> Saccharomyces cerevisiae <400> 4 atgcttcagc gaatatcatc taaacttcac aggcggttct tatctggcct gctgcgtgtc 60 aagcactacc cattaaggcg cattctcctt ccactgattc tactgcagat catcattata 120 acgtttatct ggtcaaattc accgcagcgt aacggacttg ggcgggacgc tgattacctt 180 ctaccaaatt acaacgaact tgacagtgat gatgattcct ggtatagcat cctgacttcg 240 tctttcaaaa acgatcgcaa gatccagttc gctaagacat tatacgaaaa tttaaaattc 300 ggcaccaacc ctaaatgggt caatgaatat actctgcaaa atgacctgct ctcggtcaaa 360 atgggccctc gaaagggcag taagctcgaa tccgtggatg agttgaagtt ttacgacttc 420 gaccctcgtc tcacgtggtc cgttgtgctg aaccatttgc aaaataatga cgcagatcag 480 ccagaaaagt tacccttttc atggtacgac tggacaacct tccacgagct gaataagctg 540 atttccatag ataaaactgt tctgccctgc aattttcttt tccagtccgc tttcgacaaa 600 gagtctttag aggccattga gacagagctc ggcgaacctt tgttcctata cgaaagacca 660 aagtacgcgc agaaactgtg gtacaaggcc gctagaaacc aggacagaat caaagactca 720 aaggaactaa aaaagcattg ttccaagcta ttcactccag acgggcatgg ctctcctaag 780 ggtttaagat ttaatacgca atttcaaata aaggagctgt atgataaagt tagacccgaa 840 gtttaccaat tgcaggcaag aaactacatt ttgactacac agtcgcatcc actatccatt 900 tccatcatcg aatcagataa ttccacgtat caagtcccct tgcaaactga aaaatcaaaa 960 aacttggtgc aatccggcct gttgcaggaa tatattaatg ataacattaa ttctacgaac 1020 aagagaaaga aaaataaaca ggacgtagaa ttcaaccata acaggctttt ccaggaattc 1080 gtcaataacg accaagttaa ctccctatac aaactggaaa ttgaagaaac tgataaattc 1140 acttttgata aagatttggt ttatttatcc ccttcggatt tcaagttcga tgcctccaaa 1200 aaaattgaag agttagagga acagaagaaa ctctatccgg acaaattttc cgctcataat 1260 gagaattatc tgaacagttt gaagaattcc gtaaagacaa gccctgcatt gcaaagaaag 1320 ttcttctatg aggctggtgc cgtgaagcaa tataaaggta tggggttcca tcgtgacaag 1380 aggttcttca atgttgatac attaatcaat gataaacaag aataccaggc tagattgaac 1440 tcaatgatca gaacattcca aaagtttact aaagccaacg gcatcatatc ttggttgtct 1500 cacggaacgc tgtacggcta tctttacaat ggaatggctt tcccttggga taacgatttc 1560 gacttgcaaa tgcccattaa gcatttacaa ttgctcagtc aatacttcaa ccaatctctt 1620 atattggaag acccaagaca gggtaatgga cgttatttcc tagacgtcag cgactccttg 1680 acagtaagaa ttaacggtaa cggtaaaaac aatatcgatg caagattcat tgacgtcgac 1740 accggccttt acattgatat taccggtcta gctagcactt ctgcccctag tagggattac 1800 ttgaattctt atattgaaga gcggttgcaa gaggaacatt tggatatcaa taatatccct 1860 gaatcgaacg gtgagaccgc tactttgccc gacaaagtag atgatgggtt agtcaatatg 1920 gctacactaa acatcactga gctacgtgat tacattacca gcgacgaaaa taaaaatcat 1980 aaaagagtcc ccactgatac tgatttgaaa gatcttttga aaaaggaact ggaagagtta 2040 ccaaagtcta agaccattga aaacaagttg aatcctaaac aaagatattt tctcaacgaa 2100 aaacttaaac tttacaattg tagaaacaac cattttaact cgttcgagga actatctccc 2160 ttaatcaata ctgttttcca tggtgtgcca gcgttgattc ctcacagaca tacctactgc 2220 ttgcacaatg aatatcatgt acctgataga tatgcatttg atgcttacaa aaatactgct 2280 tatttgcccg aatttagatt ttggttcgac tatgacgggt taaagaaatg cagtaatatt 2340 aattcatggt atccaaacat ccccagtatt aattcatgga atccgaacct cttgaaagaa 2400 atatcgtcta cgaaatttga gtcgaaactt tttgattcca acaaagtctc tgaatactct 2460 ttcaaaaacc tatccatgga tgatgttcgc ttaatttata aaaatattcc aaaagctggc 2520 tttatcgagg tatttactaa cttgtacaat tccttcaatg tcactgcata taggcaaaag 2580 gaattggaaa ttcaatactg ccaaaacctg acatttattg aaaaaaagaa attattacat 2640 caattgcgca ttaatgttgc tcctaagtta agctcccctg caaaggaccc atttcttttt 2700 ggttatgaaa aagctatgtg gaaggattta tcaaaatcta tgaaccagac tacattagat 2760 caagttacca agattgttca tgaagaatat gtcggaaaaa ttattgatct gtccgaaagt 2820 ttgaaataca ggaatttttc acttttcaac attacttttg atgaaactgg aacaactcta 2880 gatgataaca cagaagatta tactcctgct aatactgttg aagtaaatcc tgtggatttt 2940 aaatcaaatt taaactttag tagcaactcc tttttggatt taaattcata tggtttagac 3000 ctttttgcgc caactttatc cgacgttaac agaaagggta ttcaaatgtt tgataaggac 3060 cctattattg tatacgagga ctatgcttat gccaagttac ttgaagaaag aaagcggagg 3120 gagaagaaga agaaggagga agaggagaag aagaagaagg aagaagagga aaagaagaag 3180 aaggaagaag aagaaaagaa aaagaaggaa gaggaagaga agaaaaagaa ggaagaagaa 3240 gagaagaaaa agaaggaaga agaagaaaag aagaagcagg aggaagagga gaaaaagaag 3300 aaggaagaag aagagaagaa gaagcaggaa gaaggagaaa agatgaagaa tgaagatgaa 3360 gaaaataaga agaatgaaga tgaagaaaag aagaagaacg aagaagagga aaaaaagaag 3420 caggaagaga aaaacaagaa gaatgaagat gaagaaaaga agaagcagga agaggaagaa 3480 aagaagaaga acgaagaaga ggaaaaaaag aagcaggagg aggggcacag caat 3534 <210> 5 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Och1_pUG72_F <400> 5 atgtctagga agttgtccca cctgatcgct acaaggaaat caaaatacgc tgcaggtcga 60 caacc 65 <210> 6 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Och1_pUG72_R <400> 6 ttatttatga cctgcatttt tatcagcatc ttctttccag ctcccactag tggatctgat 60 atcacc 66 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Och1_CF <400> 7 aatggggagc gctgattctc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Och1_CR <400> 8 tctacggaag gacgttgaga 20 <210> 9 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Mnn1_pUG6_F <400> 9 aacgtaatct tgcggtattt aacgctagtt taagaaagtg ttactgtgta tacgctgcag 60 gtcgacaacc 70 <210> 10 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Mnn1_pUG6_R <400> 10 gttcacaaag gctagtacca taaacagtta gaaaaaacac tggttaatgc actagtggat 60 ctgatatcac c 71 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mnn1_CF <400> 11 atcattgcga ggtctcaatt gg 22 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Mnn1_CR <400> 12 gattagaaaa actcatcgag catcaaatg 29 <210> 13 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_F <400> 13 acaacgtcac tattccttca cacaaataaa ctaattagtt atgcttcagc gtacgctgca 60 ggtcgacaac c 71 <210> 14 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_R <400> 14 aggaaaggct atagaaatga agagattcat gaattttcag tcaggttcta ctagtggatc 60 tgatatcacc 70 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Leu2_CF <400> 15 agccttgtca agagaccaga 20 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_CR <400> 16 attgtttgcc acttatcact ggcg 24 <210> 17 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Mnn6_F <400> 17 agcgttcacc caaccttttg tgccctttag tgaagataag ataaggtaag tacgctgcag 60 gtcgacaacc 70 <210> 18 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Mnn6_R <400> 18 tatatattca tatgtagaag attattgttc ttatacatca gtgttttgat actagtggat 60 ctgatatcac c 71 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mnn6_CF <400> 19 gctctcgtga gacacgagtt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ura3_CR <400> 20 cccgtcaatt agttgcacca 20 <210> 21 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Mnn14_F <400> 21 atgatgttat cactgcgcag gttctccatg tacgttttga gatctctgcg tacgctgcag 60 gtcgacaacc 70 <210> 22 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Mnn14_R <400> 22 ttaatatttt tggtctgaac caaatatatt gtttgaagtt ttcttattat actagtggat 60 ctgatatcac c 71 <210> 23 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Yur1_F <400> 23 atggcaaaag gaggctcgct atacatcgtt ggcatattct taccaatatg tacgctgcag 60 gtcgacaacc 70 <210> 24 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Yur1_R <400> 24 ttaaatctcg tcttgctctt cttttaagaa atatttgccg ctaccgtttt actagtggat 60 ctgatatcac c 71 <210> 25 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Ktr2_F <400> 25 atgcaaatct gcaaggtatt tcttacacag gttaaaaaac tactttttgt tacgctgcag 60 gtcgacaacc 70 <210> 26 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Ktr2_R <400> 26 ctatgaatcg tgtttgagga agtatttacc gctgccgtcc ttccaccatc actagtggat 60 ctgatatcac c 71 <210> 27 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Ktr4_F <400> 27 atgaggtttc tttcaaaaag gatactgaaa cctgtacttt cagtgatcat tacgctgcag 60 gtcgacaacc 70 <210> 28 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Ktr4_R <400> 28 tcaatacatt tctaactctt cctcagacat agagtgtctt atccaggttg actagtggat 60 ctgatatcac c 71 <210> 29 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Ktr5_F <400> 29 atgttgctaa taagaaggac gataaatgca tttctgggat gtatccattg tacgctgcag 60 gtcgacaacc 70 <210> 30 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Ktr5_R <400> 30 ctagtttccg aactgtctta gatagtcttc ccttatgtgc tcctccattt actagtggat 60 ctgatatcac c 71 <210> 31 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Ktr7_F <400> 31 atggctataa gattgaatcc aaaagtcaga aggttcttgc tggataagtg tacgctgcag 60 gtcgacaacc 70 <210> 32 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Ktr7_R <400> 32 ctattcaatt actctaaaat tttctcttct gatctcttca atcacgtctt actagtggat 60 ctgatatcac c 71 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Mnn14_CF <400> 33 cgaagatcaa gtaagagtgc acttg 25 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Yur1_CF <400> 34 atctgtcact gcttattcat atcatc 26 <210> 35 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Ktr2_CF <400> 35 atctcttcag gtatgtgaca cctata 26 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Ktr4_CF <400> 36 caacggaacg agctctataa gacg 24 <210> 37 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Ktr5_CF <400> 37 acactttaag catgcggtgt gtgga 25 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Ktr7_CF <400> 38 gtatacatca ggctaacaat ctgtga 26 <210> 39 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> GAPDHp-F1 <400> 39 agtcgaattc atactagcgt tgaatgttag cg 32 <210> 40 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> GAPDHp-R1 <400> 40 agtcggtacc tttgtttgtt tatgtgtgtt tattc 35 <210> 41 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> GAPDHt-F1 <400> 41 agtcggtacc ggatcctcta gagtgaattt actttaaatc ttgcat 46 <210> 42 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> GAPDHt-R1 <400> 42 agtcctgcag atccacaatg tatcaggtat ct 32 <210> 43 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_F <400> 43 cgcggatcca tgcttcagcg aatatcatct aaac 34 <210> 44 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Mnn4_R <400> 44 ggactagttt aattgctgtg cccctcctc 29 <210> 45 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Y-Mnn14-F <400> 45 aacacacata aacaaacaaa ggtaccatga tgttatcact gcgcagg 47 <210> 46 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Y-Mnn14-R <400> 46 aaattcactc tagaggatcc ggtaccttaa tatttttggt ctgaaccaaa 50 <210> 47 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> p-Gas1-F <400> 47 tacacgtact tagtcgctga agctcttcta tgatgttgtt taaatccctt tcaaag 56 <210> 48 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> p-Gas1-R-1 <400> 48 gtgatgatga tgatggtggt ggtgagaacc ccctccacca ctggattcag ttccggaacc 60 60 <210> 49 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> p-Gas1-R-2 <400> 49 aggtacgaac tcgattgacg gctcttctac cgtgatgatg atgatggtgg tg 52
Claims (3)
A method for adding mannose phosphoric acid to a sugar chain, which comprises the step of treating a sugar chain with Mnn14 protein represented by the amino acid sequence of SEQ ID NO: 1 to add mannose phosphoric acid.
The method according to claim 1, wherein the sugar chain is located in a glycoprotein, wherein mannose phosphate is added to the oligosaccharide.
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