KR0132003B1 - Process for preparation of recombinant proteins by use of streptomyces thermonitrificans aminopeptidase - Google Patents
Process for preparation of recombinant proteins by use of streptomyces thermonitrificans aminopeptidaseInfo
- Publication number
- KR0132003B1 KR0132003B1 KR1019940010266A KR19940010266A KR0132003B1 KR 0132003 B1 KR0132003 B1 KR 0132003B1 KR 1019940010266 A KR1019940010266 A KR 1019940010266A KR 19940010266 A KR19940010266 A KR 19940010266A KR 0132003 B1 KR0132003 B1 KR 0132003B1
- Authority
- KR
- South Korea
- Prior art keywords
- aminopeptidase
- protein
- growth hormone
- human growth
- lys
- Prior art date
Links
- 102000004400 Aminopeptidases Human genes 0.000 title claims abstract description 29
- 108090000915 Aminopeptidases Proteins 0.000 title claims abstract description 28
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 15
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims description 15
- 238000002360 preparation method Methods 0.000 title claims description 4
- 241000187177 Streptomyces thermovulgaris Species 0.000 title abstract description 3
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 28
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 108010000521 Human Growth Hormone Proteins 0.000 claims description 17
- 102000002265 Human Growth Hormone Human genes 0.000 claims description 17
- 239000000854 Human Growth Hormone Substances 0.000 claims description 17
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000011535 reaction buffer Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 241000187747 Streptomyces Species 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 abstract description 8
- 239000012531 culture fluid Substances 0.000 abstract 1
- 229930182817 methionine Natural products 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 108700031632 somatrem Proteins 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 239000007983 Tris buffer Substances 0.000 description 7
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 6
- 125000000539 amino acid group Chemical group 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 206010062767 Hypophysitis Diseases 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 210000003635 pituitary gland Anatomy 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- SITWEMZOJNKJCH-WDSKDSINSA-N Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N SITWEMZOJNKJCH-WDSKDSINSA-N 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- GGLZPLKKBSSKCX-YFKPBYRVSA-N L-ethionine Chemical group CCSCC[C@H](N)C(O)=O GGLZPLKKBSSKCX-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- UASDAHIAHBRZQV-YUMQZZPRSA-N Met-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N UASDAHIAHBRZQV-YUMQZZPRSA-N 0.000 description 1
- QXOHLNCNYLGICT-YFKPBYRVSA-N Met-Gly Chemical compound CSCC[C@H](N)C(=O)NCC(O)=O QXOHLNCNYLGICT-YFKPBYRVSA-N 0.000 description 1
- IMTUWVJPCQPJEE-IUCAKERBSA-N Met-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN IMTUWVJPCQPJEE-IUCAKERBSA-N 0.000 description 1
- 108090000192 Methionyl aminopeptidases Proteins 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000607269 Vibrio proteolyticus Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 150000003931 anilides Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 125000002073 methionyl group Chemical group 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/11—Aminopeptidases (3.4.11)
- C12Y304/11002—Membrane alanyl aminopeptidase (3.4.11.2), i.e. aminopeptidase N
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Endocrinology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
제1도는 본 발명에 사용되는 아미노펩티다제의 각 pH 구간에서의 활성도를 나타낸 것이고,Figure 1 shows the activity in each pH section of the aminopeptidase used in the present invention,
제2도는 100mM 트리스 완충용액(pH 8.0) 중에서의 염화나트륨 농도 변화에 따른 상기 아미노펩티다제의 상대 활성도의 변화를 나타낸 것이고,Figure 2 shows the change in the relative activity of the aminopeptidase according to the change in sodium chloride concentration in 100mM Tris buffer (pH 8.0),
제3a 및 3b도는 각각 아미노펩티다제와의 반응에 의해 N-말단의 메티오닐이 제거되기 전과 후의 N-말단 서열 분석 결과를 나타낸 것이며,3a and 3b show N-terminal sequence analysis results before and after N-terminal methionyl is removed by reaction with aminopeptidase, respectively.
제4도는 본 발명에 따라 최종 제조된 천연형 재조합 인간 성장 호르몬을 나트륨 도데실 설페이트-폴리아크릴아미드 겔전기영동(SDS-PAGE)한 결과를 나타낸 것이다.Figure 4 shows the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the natural recombinant human growth hormone finally prepared according to the present invention.
본 발명은 유전자 재조합 방식에 의해 제조된 메티오닐 단백질로부터 아미노펩티다제(aminopeptidase)를 이용한 천연형과 동일한 아미노산 서열을 갖는 재조합 단백질을 제조하는 방법에 관한 것으로, 보다 상세하게는 x-Pro, Met-Ala, Met-Gly, Met-Lys 및 Met-Arg의 아미노산 서열을 갖는 단백질을 제외한 N-말단에 메티오닌 잔기를 갖는 단백질, 예를 들면 메티오닐 인간 성장 호르몬(methionyl human growth hormone)을 스트렙토마이세스 써모니트리피컨스(Streptomyces thermonit rificans, ATCC 23385)의 배양액으로부터 얻은 아미노 펩티다제로 처리하여 상기 단백질의 N-말단의 메티오닌을 제거함으로서 천연형 재조합 단백질을 제조하는 방법에 관한 것이다.The present invention relates to a method for preparing a recombinant protein having the same amino acid sequence as that of a native form using an aminopeptidase from a methionyl protein prepared by a gene recombination method, and more specifically, x-Pro, Met Streptomyces is a protein having a methionine residue at the N-terminus, for example methionyl human growth hormone, except for proteins having amino acid sequences of Ala, Met-Gly, Met-Lys and Met-Arg. The present invention relates to a method for producing a native recombinant protein by removing an N-terminal methionine of the protein by treatment with an amino peptidase obtained from a culture solution of Streptomyces thermonit rificans (ATCC 23385).
인체로부터 유래되는 단백질은 그 양이 매우 제한될 뿐 아니라 안정성에 있어서도 많은 문제가 제기되고 있다. 예를 들어, 뇌하수체 유래 인간성장호르몬을 접종받은 어린이 중 4명이 바이러스 감염에 의해 사망한 것으로 밝혀짐으로써 미국 식품 의약국으로부터 이의 사용이 금지되기도 하였다[Science. 234, 22(1986)].Proteins derived from the human body are not only limited in amount but also pose many problems in stability. For example, four children out of pituitary gland-derived human growth hormone have been found to have died from viral infections, and their use has been banned by the US Food and Drug Administration [Science. 234, 22 (1986).
따라서, 최근에는 유전공학 기술을 이용하여 대장균 및 효모에서 인체 유래 단백질을 발현 및 분리 정제하여 임상에 이용하고 있다. (대장균:대한민국 특허공고 제89-1244 및 87-701 호 및 특허공개 제 87-2258 및 84-8695호 :효모:대한민국 특허공고 제 92-99호 및 특허공개 제 90-9973 및 90-9976호 참조)Therefore, recently, human-derived proteins have been expressed and isolated and purified from E. coli and yeast using genetic engineering techniques. (E. coli: Korean Patent Publication Nos. 89-1244 and 87-701 and Patent Publication Nos. 87-2258 and 84-8695: Yeast: Korean Patent Publication Nos. 92-99 and 90-9973 and 90-9976 Reference)
그러나 상기 방법으로 재조합 인체 유래 단백질을 생산할 경우, 천연형 단백질을 구성하는 아미노산 잔기 이외에 N-말단에 핵산전사 개시 코돈(codon)인 ATG에서 전이된 메티오닌 전기가 하나 더 부가된 아미노산 잔기로 이루어진 메티오닐 단백질이 생성된다. 이러한 현상은 재조합 인간 성장 호르몬을 제조할 때 보편적으로 이용되는 숙주인 대장균 또는 효모에서 공히 나타나는 결과이다.However, when producing a recombinant human-derived protein by the above method, in addition to the amino acid residues constituting the native protein, methionyl consisting of an amino acid residue additionally added to the methionine electricity transferred from ATG, the nucleic acid transcription initiation codon at the N-terminus Protein is produced. This phenomenon is the result of both E. coli or yeast, a host commonly used in the production of recombinant human growth hormone.
예를들어, 천연형 인간성장 호르몬은 191개의 아미노산 잔기로 이루어지는 반면 재조합 메티오닐 인간 성장 호르몬은 192개의 아미노산 잔기로 이루어지나 생물학적 역가면에서는 천연형 인간 성장 호르몬과 동일한 성질을 나타내고[Moore. J. A. Endocrinology, 122:2920-2926 (1988)], 또한 메티오닌 잔기가 첨가됨으로 인해 발생하는 부작용 등은 보고된 바 없으나, 첨가된 N-말단 메티오닌으로 인하여 간혹 항체가 생성될 수가 있어서 항체 생성율이 천연형보다 약간 높다는 보고가 있었다[Lacer. March 29, 697, (1996)].For example, native human growth hormone consists of 191 amino acid residues, whereas recombinant methionyl human growth hormone consists of 192 amino acid residues, but in biological titers it exhibits the same properties as native human growth hormone [Moore. JA Endocrinology, 122: 2920-2926 (1988)], and also side effects due to the addition of methionine residues have not been reported, but the antibody production rate is natural because the added N-terminal methionine can sometimes produce antibodies. It was reported to be slightly higher than [Lacer. March 29, 697, (1996).
따라서, 재조합 인간 성장 호르몬 등의 재조합 인체 유래단백질의 제조시 N-말단에 메티오닌이 없는 천연형 재조합 인간 성장 호르몬을 제조하고자 하는 많은 시도가 있었다. 예를 들면, 인간 성장 호르몬의 N-말단과 다른 단백질의 C-말단을 융합 발현시킨 후 특수한 프로테아제(protease)를 이용하여 절단하는 방법(PCT 국제 출원공개 제 WO 89/12678호, EP 제 20290호, EP 제 321940호), 단백질을 세포내에서 발현시켜 숙주세포 밖으로 분비될 때 메티오닌이 절단 제거되도록 함으로써 배지로부터 천연형 단백질을 수득하는 방법 등이 있다 (유럽 특허 공개 제 0088632 A2호, 미국 특허 제 4755465호, 일본 특허 제 01273591호, Ep 제 306673호, 대한민국 특허출원 제 92-10932).Therefore, there have been many attempts to produce naturally occurring recombinant human growth hormone without methionine at the N-terminus in the production of recombinant human derived proteins such as recombinant human growth hormone. For example, a method of fusion expression of the N-terminus of human growth hormone and the C-terminus of another protein followed by cleavage using a special protease (PCT International Publication No. WO 89/12678, EP 20290) (EP 321940), to obtain a native protein from the medium by expressing the protein intracellularly, so that methionine is cleaved off when secreted out of the host cell (European Patent Publication No. 0088632 A2, US Patent No. 4755465, Japanese Patent No. 01273591, Ep No. 306673, Korean Patent Application No. 92-10932).
상기의 방법들은 새로 벡터를 만들어 숙주세포로 형질전환하는 등의 번거로운 조작과 발효 조건을 최적화시키는 노력이 부가적으로 필요하다는 단점이 있다. 이러한 부가적인 노력없어 기존에 개발된 벡터 및 숙주 세포를 이용하여 제조, 정제된 메티오닐 인체 유래 반백질을 N-말단에 존재하는 메티오닌만을 선택적으로 제거할 수 있는 특수한 아미노펩티다제로 처리하여 천연형 재조합 단백질을 제조할 수 있다(PCT 국제 출원 공개 제 WO 86/04609 및 WO 86/204527 Al 호).The above methods have the disadvantage that additional efforts for optimizing fermentation conditions and cumbersome manipulations such as transforming into host cells by newly generating vectors are required. Without this additional effort, the methionyl human-derived protein produced and purified using previously developed vectors and host cells is treated with a special aminopeptidase that can selectively remove only methionine present at the N-terminus. Recombinant proteins can be prepared (PCT International Application Publication Nos. WO 86/04609 and WO 86/204527 Al).
예를 들어, 메티오닐 인간 성장 호르몬은 그의 N-말단아미노산 서열이 Met-Phe-Pro-Thr-Ile으로 시작되므로 N-말단메티오닌만을 선택적으로 제거하기 위해 사용될 수 있는 아미노펩티다제는, N-말단의 X-Pro(X는 임의의 아미노산 잔기)를 인지하여 절단 반응이 X 잔기앞에서 정지되는 특성을 지니고 있는 효소이어야 하며, 그 가운데서도 효소 활성도가 높은 것일수록 산업적으로 이용하는데 유리하다.For example, methionyl human growth hormone is an aminopeptidase that can be used to selectively remove only N-terminal methionine since its N-terminal amino acid sequence begins with Met-Phe-Pro-Thr-Ile, N- It should be an enzyme having the characteristic that the cleavage reaction is stopped in front of the X residue by recognizing the terminal X-Pro (X is any amino acid residue), and the higher the enzyme activity, the more advantageous for industrial use.
현재까지 알려진 N-말단 메티오닌 잔기 제거에 의한 천연형 재조합 단백질의 제조에 이용되는 아미노 펩티다제로는 비브리오 프로테오라이티커스(Vibrio proteolyticus)로부터 추출, 정제한 아미노펩티다제(WO 86/01229, B.T.G), 돼지 신장으로부터 추출, 정제한 아미노펩티다제(WO 86/204527 Al, 다케다(Takeda))등이 있다.The amino peptidase used to prepare a native recombinant protein by removal of N-terminal methionine residues known to date is aminopeptidase extracted and purified from Vibrio proteolyticus (WO 86/01229, BTG) and aminopeptidase (WO 86/204527 Al, Takeda) extracted and purified from porcine kidney.
본 출원인도 스트렙토마이세스 써모니트리피컨스로부터 상기 천연형 재조합 단백질의 제조에 유용한 아미노펩티다제를 분리한 바있다.(대한민국 특허출원 제 93-11107호, 명칭:신규한 아미노펩티다제 및 그의 분리방법).The present applicant has also isolated aminopeptidase useful for the production of the above-mentioned recombinant protein from Streptomyces thermoptricus. (Korean Patent Application No. 93-11107, Name: New aminopeptidase and its Separation method).
일반적으로 효소의 활성은 완충요액의 조건에 따라 가변적일 수 있기 때문에 새로운 효소의 사용시 높은 활성을 나타내도록 하기 위해서는 완충용액 조성 등 반응조건의 최적화가 필요하다.In general, since the activity of the enzyme may vary depending on the conditions of the buffer solution, it is necessary to optimize the reaction conditions such as the composition of the buffer solution in order to exhibit high activity when using a new enzyme.
본 발명자들은 상기에 언급한 스트렙토마이세스 써모니트리피컨스에서 분리 정제한 암노펩티다제를 천연형 재조합 단백질의 제조에 이용하기 위하여 완충용액의 최적 조건을 찾던 중, 특정 pH 및 염화나트륨 농도 범위에서 효소 활성이 상대적으로 증가됨을 발견하여 본 발명을 완성하게 되었다.The inventors of the present invention, while searching for the optimal conditions of the buffer solution for the use of the above-described ammopeptidase isolated from Streptomyces thermonucleus for the preparation of the native recombinant protein, the enzyme at a specific pH and sodium chloride concentration range The discovery of relatively increased activity led to the completion of the present invention.
따라서, 본 발명의 목적은 재조합 방식으로 제조된 메티오닐 단백질로부터 아미노펩티다제를 이용하여 천연형 재조합 단백질을 제조하는 방법을 제공하는 것이다. 즉, 본 발명의 목적은 N-말단에티오닌 잔기를 부가적으로 갖는 단백질을 N-말단의 메티오닌 잔기만을 선택적으로 제거하기 위한 최적 반응 조건하에서 스트렙토마이세스 써모니트리피컨스로부터 분리, 정제하여 얻은 아미노펩티다제로 처리하여 천연형 재조합 단백질을 제조하는 방법을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a method for preparing a native recombinant protein using an aminopeptidase from a methionyl protein produced in a recombinant manner. In other words, an object of the present invention is obtained by separating and purifying a protein additionally having an N-terminal ethionine residue from Streptomyces thermotropicus under optimal reaction conditions for selectively removing only N-terminal methionine residues. It is to provide a method for producing a native recombinant protein by treatment with aminopeptidase.
이하 본 발명을 상세히 설명하면 다음과 같다Hereinafter, the present invention will be described in detail.
본 발명에 따라서, N-말단에 메티오닌 잔기를 갖는 단백질, 예를 들면 메티오닐 인간 성장 호르몬을 반응 완충용액에서 최적 반응조건하에서 스트렙토마이세스 써모니트리피컨스로부터 분리된 아미노펩티다제로 처리하여 천연형 재조합 단백질을 제조하는 방법이 제공된다.According to the present invention, a protein having a methionine residue at the N-terminus, for example methionyl human growth hormone, is treated with an aminopeptidase isolated from Streptomyces thermotropicus under optimal reaction conditions in a reaction buffer solution. A method of making a recombinant protein is provided.
상기 메티오닐 단백질로는 통상의 정제 방법에 의해 대장균, 효모 등의 숙주세포로부터 얻어진 모든 메티오닐 단백질을 사용할 수 있으며, 사용하는 단백질의 분자량에 따라 아미노펩티다제의 양이 달라지나 메티오닐 인간 성장 호르몬의 경우 1mg당 0.2 내지 20유니트의 아미노펩티다제를 사용하여 처리하는 것이 바람직하다.As the methionyl protein, all methionyl proteins obtained from host cells such as E. coli and yeast can be used by a conventional purification method, and the amount of aminopeptidase varies depending on the molecular weight of the protein used, but methionyl human growth For hormones, treatment with 0.2 to 20 units of aminopeptidase per mg is preferred.
본 발명에 따르면, 반응 완충용액의 pH는 7.5 내지 9.5가 바람직하며, 가장 바람직하게는 8 내지 8.5 이고, 염화나트륨을 첨가할 수 있다. 첨가되는 염화나트륨은 최종 농도에는 특별한 제한이 없으나, 최종 농도가 0 내지 100mM이 되게 하는 것이 바람직하다.According to the present invention, the pH of the reaction buffer is preferably 7.5 to 9.5, most preferably 8 to 8.5, and sodium chloride may be added. The sodium chloride added is not particularly limited in the final concentration, but it is preferable to bring the final concentration to 0 to 100 mM.
따라서, 본 발명은 메티오닐 단백질, 특히 메티오닐 인간 성장 호르몬을 염화나트륨이 0내지 100mM의 최종농도로 첨가된 pH 7.5 내지 9.5의 반응 완충용액중에서 스트렙토마이세스 써모니트리피컨스로부터 분리된 아미노펩티다제로 처리함으로써 효소 활성에 최적인 반응 조건하에 37℃에서 N-말단의 메티오닌 잔기만을 선택적으로 제거하는 천연형 재조합 단백질의 제조방법을 제공한다.Accordingly, the present invention relates to a methionyl protein, particularly methionyl human growth hormone, as an aminopeptidase isolated from Streptomyces thermotropicus in a reaction buffer of pH 7.5 to 9.5 with sodium chloride added at a final concentration of 0 to 100 mM. The present invention provides a method for producing a native recombinant protein which selectively removes only N-terminal methionine residues at 37 ° C. under reaction conditions optimal for enzymatic activity.
본 원에서는 재조합 단백질의 예로서 메티오닐 인간 성장 호르몬을 들어 기술하였으나, 본 발명의 방법은 다른 단백질에도 응용가능함은 물론이다.Although herein described methionyl human growth hormone as an example of a recombinant protein, the method of the present invention is of course applicable to other proteins as well.
이하 본 발명을 실시예에 의거하여 보다 상세히 설명한다. 실시예에서 사용되는 메티오닐 인간 성장 호르몬은 대한민국 특허 출원 제 90-3465호에 의해 명시된 방법으로 정제된 것이고, 아미노펩티다제로는 대한민국 특허출원 제 93-11107호에 명시된 스트렙토마이세스 써모니트리피컨스에서 분리된 아미노펩티다제를 사용하였다.Hereinafter, the present invention will be described in more detail with reference to Examples. The methionyl human growth hormone used in the examples was purified by the method specified by Korean Patent Application No. 90-3465, and as the aminopeptidase, the Streptomyces Thermotripicus specified in Korean Patent Application No. 93-11107 Aminopeptidase isolated at was used.
아미노펩티다제의 유니트는 100mM의 트리스(pH 8.0)에 용해된 류신-파라니트로 아닐리드(Leucine-PNA)를 기질로 한 완충용액(1㎖)에 아미노펩티다제를 가하여 37℃에서 반응시켰을 때 405nm에서 분당 OD 1만큼을 변화시키는 효소의 양으로 정의된다.The unit of aminopeptidase was reacted at 37 ° C by adding aminopeptidase to a buffer solution (1 ml) based on leucine-paranitro anilide (Leucine-PNA) dissolved in 100 mM Tris (pH 8.0). It is defined as the amount of enzyme that changes OD 1 per minute at 405 nm.
하기의 실시예는 본 발명을 보다 구체적으로 설명하기 위해 예시된 것일 뿐이고 본 발명이 이로 한정되는 것은 아니다.The following examples are only illustrated to illustrate the present invention in more detail, but the present invention is not limited thereto.
참조예 반응 완충용액의 최적 조건의 결정Reference Example Determination of Optimal Conditions for the Reaction Buffer
반응 완충용액의 최적조건은 본 출원인이 동일자로 출원한 대하민국 특허출원 제_______호의 방법을 이용하여 조사하였다. 기질로서 메티오닐펩티드(Met-Phe-Pro-Thr-Glu-Pro-Ser)를 포함하는 0.1M 트리스 완충용액을 각각 pH 7, pH 7.5, pH 8, pH 8.5 및 pH9의 pH 별로 제조하고, 상기 기질을 포함하는 인산 나트륨 완충용액을 pH 6에서 pH 7.5까지 pH 0.5간격으로 제조하고, 중탄산 나트륨 완충용액을 pH 8.5 내지 pH 10까지 0.5의 pH 간격으로 pH 별로 제조한 후 각 완충용액 1㎖에 동량의 아미노펩티다제를 첨가하여 10분간 처리한 후 그 결과를 제1도에 나타내었다. 이로부터, pH 7.5 내지 9.5의 완충용액에서의 처리가 다른 완충용액에서 보다 상대적으로 우수한 효소 활성을 나타냄을 알 수 있었다.Optimal conditions of the reaction buffer solution were investigated using the method of Patent Application No. _______ of the Republic of Korea filed by the same applicant. 0.1M Tris buffer containing methionyl peptide (Met-Phe-Pro-Thr-Glu-Pro-Ser) as a substrate was prepared for each pH of pH 7, pH 7.5, pH 8, pH 8.5 and pH9, and Sodium phosphate buffer solution containing the substrate was prepared at a pH of 0.5 to pH 7.5, and sodium bicarbonate buffer solution was prepared for each pH at a pH interval of 0.5 to pH 8.5 to pH 10, and then the same amount in each 1 ml of buffer. After treatment for 10 minutes with the addition of the aminopeptidase of the results are shown in FIG. From this, it can be seen that the treatment in the buffer solution of pH 7.5 to 9.5 shows a relatively better enzyme activity in the other buffer solution.
또한 적절한 이온 강도를 유지하여 단백질의 안정성 감소를 방지하기 위해 염화나트륨을 각각 0,100,200,300,400 및 500mM의 최종농도로 첨가한 100mM 트리스 완충요액(pH 8.0)중에서 상기 기질을 효소처리하고 그 결과를 제2도에 나타내었다. 도면에서 알 수 있듯이, 염화나트륨의 농도가 증가함에 따라서 효소 활성도가 상대적으로 감소하였다.In addition, the substrate was enzymatically treated in 100 mM Tris buffer solution (pH 8.0) in which sodium chloride was added to final concentrations of 0,100,200,300,400 and 500 mM, respectively, in order to maintain the proper ionic strength and prevent the reduction of protein stability. It was. As can be seen from the figure, the enzyme activity was relatively decreased as the concentration of sodium chloride increased.
실시예 1 : 천연형 재조합 인간 성장 호르몬의 제조Example 1 Preparation of Natural Recombinant Human Growth Hormone
약 600㎍의 메티오닐 인간 성장 호르몬에 아미노펩티다제 8유니트를 첨가하고 반응완충용액(50mM 트리스 (pH 8.0), 100mM NaCl, 및 1mM PMSF)으로 충분히 투석시켜 준 다음, 2㎖ 용량의 세트리콘(centricon, 아미콘(Amicon)사)을 이용하여 부피를 0.6㎖까지 조절한 후 37℃의 물 중탕기에 24시간동안 담가 놓았다. 약 20시간이 경과한 후 동일한 반응 완충용액으로 재투석하였다.Add 8 units of aminopeptidase to about 600 μg of methionyl human growth hormone and allow sufficient dialysis with reaction buffer solution (50 mM Tris (pH 8.0), 100 mM NaCl, and 1 mM PMSF), followed by 2 ml of setricone. (centricon, Amicon) was used to adjust the volume to 0.6ml and soak for 24 hours in a water bath at 37 ℃. After about 20 hours it was redialysis with the same reaction buffer.
상기 반응이 완료되면, 이 반응액을 20mM 트리스 완충용액 (pH 8.0)으로 충분히 투석시킨 후, 동일 완충용액으로 미리 평형시킨 DEAE-세파로즈 칼럼에 적용하고, 동일 완충용액으로 세척한 다음 20mM트리스, 100mM NaCl 완충용액으로 인간 성장 호르몬을 용출하였다.Upon completion of the reaction, the reaction solution was sufficiently dialyzed with 20 mM Tris buffer (pH 8.0), applied to a DEAE-Sepharose column previously equilibrated with the same buffer, washed with the same buffer and then 20 mM tris, Human growth hormone was eluted with 100 mM NaCl buffer.
실시예 2 : 천연형 재조합 인간 성장 호르몬의 특성조사Example 2 Characterization of Natural Recombinant Human Growth Hormone
실시예 1에서 얻어진 N-말단 메티오닌 잔기가 제거된 천연형 재조합 인간 성장 호르몬을 N-말단 서열 분석기(어플라이드 바이오시스템즈 471A 단백질 서열 분석기(Applied Biosystems 471A Protein Sequencer)를 사용하여 메티오닐기가 제거된 정도를 확인하고 그 결과를 제3도에 나타내었다. 제3a 도는 N-말단의 메티오닌이 제거되기 전, 제3b도는 아미노펩티다제 처리 후의 N-말단 서열 분석 결과를 나타낸다. 도면에서 보면 인간 성장 호르몬의 첫번째 아미노산은 페닐알라닌으로 나타났으며 메티오닌 위치에서는 거의 피크가 보이지 않았다.The degree of methionyl group removal using the N-terminal sequencing analyzer (Applied Biosystems 471A Protein Sequencer) was determined using the native recombinant human growth hormone obtained by removing the N-terminal methionine residue obtained in Example 1. The results are shown in Figure 3. Figure 3a shows the results of N-terminal sequence analysis after aminopeptidase treatment before the N-terminal methionine is removed. The first amino acid appeared as phenylalanine and showed little peak at the methionine position.
따라서, 본 발명에 따른 상기 처리에 의해서 N-말단의 메티오닌 잔기가 거의 완전히 제거되었으며, 또한 N-말단의 메티오닌만이 선택적으로 절단되고 더 이상의 아미노산은 절단되지 않음이 확인되었다.Thus, it was confirmed that the treatment according to the present invention almost completely eliminated the N-terminal methionine residues, and that only the N-terminal methionine was selectively cleaved and no further amino acids were cleaved.
N-말단의 메티오닌이 제거된 천연형 재조합 인간 성장 호르몬으 순도 측정은 SDS-PAGE를 이용하여 수행하였으며, 결과는 제4도의 2레인에 나타나 있다.Purity measurement of native recombinant human growth hormone with N-terminal methionine removed was performed using SDS-PAGE, and the results are shown in lane 2 of FIG.
최종적으로 정제된 천연형 재조합 인간 성장 호르몬의 역가를 방사 수용체 분석법(radio receptor assay)으로 측정한 결과, 세계보건기구(WHO)에서 공급하는 뇌하수체 유래 인간 성장 호르몬의 역가인 2.5IU/mg에 준하는 2.7IU/mg으로 나타났다(대한 내분비 학회지 5권 제3호, 1990).Finally, the titer of purified natural recombinant human growth hormone was measured by a radio receptor assay, and 2.7 equivalent to 2.5 IU / mg, the titer of pituitary gland derived human growth hormone supplied by the World Health Organization (WHO). IU / mg (Korean Endocrinology Vol. 5, No. 3, 1990).
이상의 설명에서 알 수 있는 바와 같이, 본 발명자들에 의해 고안된 메티오닐 인간 성장 호르몬과 아미노펩티다제와의 반응조건은 천연형 재조합 인간 성장 호르몬의 물리화학적 성질을 그대로 유지시키면서 N-말단의 메티오닌만을 선택적으로 제거하는 최적의 반응 조건임이 입증되었다.As can be seen from the above description, the reaction conditions of the methionyl human growth hormone and aminopeptidase devised by the present inventors are only N-terminal methionine while maintaining the physicochemical properties of the native recombinant human growth hormone. It has proved to be the optimal reaction condition to selectively remove.
이상과 같이, 본 발명을 구체적으로 실시태양과 관련하여 설명하였으나, 본 발명의 범위를 벗어나지 않고 당해분야에 통상적인 지식을 가진 자에게 자명한 변경, 변형 및 수정은 본 발명의 범위에 속하는 것으로 이해하여야 한다.As described above, the present invention has been described in detail with reference to embodiments, but it will be understood that changes, modifications, and modifications apparent to those skilled in the art without departing from the scope of the present invention fall within the scope of the present invention. shall.
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