WO2015126077A1 - 안정동위원소 표지 단백질 생산, 정제 및 분석 방법 - Google Patents

안정동위원소 표지 단백질 생산, 정제 및 분석 방법 Download PDF

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WO2015126077A1
WO2015126077A1 PCT/KR2015/001063 KR2015001063W WO2015126077A1 WO 2015126077 A1 WO2015126077 A1 WO 2015126077A1 KR 2015001063 W KR2015001063 W KR 2015001063W WO 2015126077 A1 WO2015126077 A1 WO 2015126077A1
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protein
coli
stable
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isotopically labeled
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최준혁
김숙경
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Korea Research Institute of Standards and Science
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

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  • the present invention relates to intracellular production, extraction, and purification of stable isotopically labeled proteins. More specifically, stable isotopically labeled proteins that can be used as internal standards for the quantitative analysis of proteins in E. coli systems. It is about the purification method and the analysis method of purified protein which can maximize the solubility and obtain high yield in the process of mass expression and extraction.
  • Human growth hormone is a single-chain polypeptide synthesized in the pituitary gland with 191 amino acid residues. Human growth hormone is known to have a variety of biological functions, including cell proliferation and metabolism. It is one of the most important hormones in the human body (Annu. Rev. Physiol., 47, 1985, 483-499) . Human growth hormone is a synthetic process agent of recombinant protein and is known as somatropin.
  • isotopically labeled proteins were synthesized using stable isotopically labeled amino acids and developed as internal standards for the quantitative analysis of target proteins.
  • the greatest advantage of this approach is that the internal standard is very similar structurally and chemically to the target protein.
  • the present inventors have used an Escherichia coli expression system to optimize the production process for isotopically labeled human growth hormone. The present invention has been completed by confirming the process. ⁇
  • [12] relates to the production of stable isotopically labeled proteins, including
  • the target protein of step (1) is not limited,
  • Escherichia coli in step (1) above can be used, but not limited to, E. coli BL21 strain, and can be cultured at 37 ° C until the OD 600 value is between 0.4 and 8.
  • Culture media also include, but are not limited to, 5 to 15 g / L Bacto Tryptone, 3 to 8 g / L yeast extract and 5 to 15 containing 40 to 60 mg / mL kanamycin.
  • Fresh LB (Luria-Breta i) medium with g / L sodium chloride can be used.
  • the stable isotope of step (5) above may be, but not limited to, "C 6 ⁇ N 4 -arginine (Argine).
  • the target protein of step (1) is not limited,
  • the stable isotopically labeled amino acid is not limited, but i3 C 6 15 N 4
  • It may be arginine.
  • the above-mentioned tablets of stable isotope marker proteins are not limited.
  • affinity chromatography and anion-exchange chromatography can be used, preferably the column of the affinity chromatography is Ni-NTA column, and the column of the anion-exchange chromatography is Mono Q column. Available have.
  • the lysis buffer solution of step (7) is not limited but 0.3 to 0.8 mM EDTA, 0.5 to L5 mg / mL lysozyme, lx protease inhibitor cocktail and It may be Tris-HCl, pH 7.5 to 8.5, including a detergent.
  • the nonionic denaturant is preferably 0.01 to 2% (v / v) Triton X-100 or Tween. May be 20).
  • the dissolved complete solution of step (7) may include, but is not limited to, salts, and preferably, the salt may be sodium chloride or potassium chloride.
  • the MALDI-TOF MS in the present invention is not limited, but may range from 600 m to 3,500 m / z, and the column temperature of the HPLC is not limited to 20 to 50. ° C, flow rate can be ⁇ 1-1.0 mL / min.
  • the present invention relates to a stable isotope labeled in the process of transforming Escherichia coli using an expression vector that encodes a protein of interest to produce a stable isotope marker hGH in the E. coli system.
  • Specific amino acids To induce the mass expression of isotopically labeled hGH, to maximize the solubility of hGH in the extraction process, to refine it to very high purity, and to analyze the isotopically labeled hGH.
  • Highly efficient expression of stable isotopically labeled proteins which can be used as internal standards for analysis, maximizes solubility during protein extraction, resulting in high yields.Affinity chromatography and ion-exchange chromatography can then be used to obtain high yields. It was possible to purify high purity hGH and based on this, a large amount of high purity
  • SI-His-hGH Stable isotopic labeling SI-His-hGH was produced, and MALDI-TOF MS was used to confirm the isotopic labeling.
  • M protein marker
  • U transformed and unexpressed cells
  • I transformed and expressed cells
  • Empty transformed cells
  • hGH non-tagged -hGH expressing cells
  • His- hGH histidine marker -hGH expressing cells.
  • FIG. 2 shows the purification of His-hGH in non-optimal native conditions.
  • FIG. 3 shows the purification of His-hGH under optimized native conditions.
  • FIG. 5 shows the primary purification of SI-His-hGH under optimized native conditions.
  • FIG. 6 shows a peptide predicted after trypsinizing His-hGH.
  • FIG. 7 is a diagram illustrating mass measurement values of four representative peptides generated after trypsin treatment of the positive control LG-hGH and SI-His-hGH using a MALDI-TOF mass spectrometer.
  • FIG. 8 is a graph comparing the Lift TOF TOF (MS / MS) spectrum of the LG-hGH trypsin peptide of m / z 1205.574 with the spectrum of SI-His-hGH peptide of m / z 1215.429.
  • 9 shows ion-exchange chromatography of His-hGH using a monocube column. 10 shows the results of silver staining analysis of the monocube fraction.
  • NM_000515.3: 141-719) requested the synthesis of BIONIA (BIONEER, Daejeon, Korea), Nhel restriction enzyme cleavage site (GCTAGC) at the 5 'end and Xhol restriction enzyme cleavage site (CTCGAG) at the 3' end.
  • BIONIA BIONIA
  • GCTAGC Nhel restriction enzyme cleavage site
  • CTCGAG Xhol restriction enzyme cleavage site
  • the synthesized cDNA was ligation to pET-28a (Novagen, Madison, WI, USA) using restriction enzymes Nhel and Xh to cut 6-histidine label and truncated bin at the N-terminus.
  • a His-hGH expression vector expressing a recombinant human growth hormone with site was completed.
  • Three His-hGH presenting combination DNAs were transformed into Escherichia coli BL21 (DE3).
  • the E. coli was inoculated into a 250 mL flask containing 50 mg / mL kanamycin and incubated overnight at 37 ° C. 5 mL of the culture medium was centrifuged at 10,000 xg for 5 minutes, followed by M9 medium containing 0.2 wt% glucose (Cat No.
  • the treated culture was inoculated into a 4L flask containing 500 mL of M9 medium containing kanamycin and incubated at 37 ° C until a value of OD 600 of about 0.6 was obtained.
  • a 25 X amino acid complex was prepared by adding 19 amino acids lg, except arginine (Arg), to 200 mL distilled water, followed by "C 15 N-arginine (Arg) labeled with radioisotopes in 1 mL distilled water.
  • a solution containing 250 g was prepared: 20 mL of 25 X amino acid complex and 13 C 15 at a concentration of 250 mg / mL.
  • N-arginine (Arg) 400 was added and incubated for 30 minutes. The solution ().
  • coli cells were disrupted with 25 mL of lysate buffer solution (1 mg / mL lysozyme, 1 X protease inhibitor cocktail (Rosh, Spain) and 50 mM Tris-HCl containing 0.5 mM EDTA) and then ultrasonically at 10,000 xg. Centrifuged for 20 minutes to obtain insoluble and soluble fractions
  • Fig. 1 it can be seen that His-hGH is expressed by IPTG, which induces protein synthesis. From the above results, His-hGH expressing DNA was selected as a target of the following examples.
  • E. coli BL21 (DE3), which expresses human growth hormone (His-hGH and SI-ffis-hGH), was cultured in 250 mL culture medium and harvested for 16 hours at 16 ° C. The harvested cells were dissolved in 25 mL lysate layer solution (pH 8.0 50 mM Tris-HCl) containing 0.5 mM EDTA, 0.1% Triton X-100, 1 mg / mL lysozyme and 1 X Protease Inhibitor Cocktail (Rosh, Spain). After sonication, centrifugation was performed at 10,000 xg for 20 minutes to obtain and purify.
  • lysate layer solution pH 8.0 50 mM Tris-HCl
  • Triton X-100 0.1% Triton X-100
  • 1 mg / mL lysozyme 1 X Protease Inhibitor Cocktail
  • Each column was infused with agarose beads hnL (Qiagen, Valencia, CA, USA).
  • the His-hGH and SI-His-hGH proteins injected into the column were washed with the wash buffer solution of Table 2, which was three times the volume of the column, eluted with 10 mL elution buffer of Table 2, and then His-hGH and SI.
  • -His-hGH was first purified. His-hGH and
  • PBS Phosphate buffered saline
  • Dialysis fractions are 5/50 monocuum columns, anion-exchange chromatography (GE)
  • the purified protein obtained in the Examples was stained with Coomassie stained gel.
  • Pretreatment for MALDI-TOF MS was performed using an in-gel digest method, in which the target protein was separated from the gel to treat trypsin. After cutting off each of the gel fragments containing His-hGH and SI-His-hGH, induction of reduction and alkylation is incubated for 10 minutes at 66 ° C. Trypsin is 25 mM ammonium bicarbonate (ammonium) at a concentration of 22.5 ng ⁇ L. Dissolved in bicarbonate, NH 4 HC0 3 ), gel fragments and reaction at 37 ° C overnight to induce peptide formation. The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) was induced. 1 mg / mL of the obtained protein was analyzed using MALDI matrix.
  • MALDI-TOF matrix-assisted laser desorption / ionization time-
  • Trifluoroacatic acid (TFA) in water) and B (0.1% Trifluoroacatic acid (TFA) in acetonitrile (ACN)) were used, and the linear gradient of elution layer B was changed from 35% to 100% for 7 min. o eluted from C. the flow rate was 0.4 mL / min, to measure the UV absorption at 220 nm wavelength.
  • Figure 7 shows the mass control peptides generated after trypsin treatment with LG-hGH and SI-His-hGH in the control group using a MALDI-TOF mass spectrometer.
  • FIG. 9 after the ion-exchange chromatography was performed for the secondary purification of SI-His-hGH, the result was shown.
  • SI-His-hGH purified purely from the fractions was identified by silver staining, and it was confirmed that it was well separated from other impurities.
  • the fraction of SI-His-hGH identified in FIG. 10 was analyzed by HPLC. 11 is shown. As a result of analyzing the positive control group LG-hGH and SI-His-hGH, it was confirmed that the purified SI-His-hGH had a high level of purity.
  • sequence listing was attached as a sequence listing file.

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Abstract

본 발명은 안정동위원소 표지 단백질의세포내생산및추출,정제방법에 관한 것이다. 보다 상세하게는 대장균 시스템에서 단백질을 대량발현시,목적단백질의추출 과정에서 용해도를 최대화하며, 생물학적 활성을 가지고, 다량의 수득률로 얻을 수 있는 정제방법 및 이의 분석에 관한 것이다.본 발명을 통해 대량의 고순도 hGH를 정제하였고, 안정동위원소 표지 SI-His-hGH를 합성하여 표지 여부를 성공적으로 확인하였다.

Description

명세서
발명의명칭:안정동위원소표지단백질생산，정제및분석방법 기술분야
[1] 본발명은안정동위원소표지단백질의세포내생산및추출，정제방법에관한 것이다.보다상세하게는단백질의정량분석을위한내부표준물질로이용할수 있는안정동위원소표지단백질을대장균시스템에서대량발현하고추출하는 과정에서용해도를최대화하여높은수득를로얻을수있는정제방법및정제된 단백질의분석법에관한것이다.
배경기술
[2] 인간성장호르몬 (human growth hormone, hGH)은 191개의아미노산잔기를 가지며뇌하수체에서합성되는단일사슬폴리펩타이드이다.인간성장호르몬은 세포증식과신진대사를포함한다양한생물학적기능을하는것으로알려져 있으며，인체에가장중요한호르몬중하나이다 (Annu. Rev. Physiol., 47, 1985, 483-499).인간성장호르몬은재조합단백질의합성과정제가가능하며 소마트로핀 (somatropin)이라하여인체내인간성장호르몬인
소마토트로핀 (somatotropin)과구별된명칭을사용한다.
[3] 단백질정량에관한최근연구는생물학적으로복잡한시료에서표적단백질을 절대적으로정량하기위한기술개발에초점이맞추어져있다.이를위해내부 표준물질 (internal standard)로안정동위원소 (stable isotope)가표지된
펩타이드 (peptide)를이용하여펩타이드수준으로절단된표적단백질의양을 유추하는실험원리가많이이용되고있다.그러나，이러한방법은고비용의 펩타이드합성이선행되어야하고，표적단백질의절단효율이정량분석에큰 영향을미치는단점으로보다나은대안이필요한실정이다.
발명의상세한설명
기술적과제
[4] 상기와같은문제점을해결하기위해재조합 DNA기술 (recombinant DNA
technology)과안정동위원소표지아미노산을이용하여동위원소표자단백질을 합성하고，이를표적단백질의정량분석을위한내부표준물질로개발하였다. 상기접근법의최대장점은내부표준물질이표적단백질과구조적，화학적으로 매우유사하다는점이다.이에본발명자들은대장균 (Escherichia coli)발현 시스템을이용하여동위원소표지된인간성장호르몬의최적화된생산과정제 과정을확인함으로써본발명을완성하였다. ᅳ
과제해결수단
[5] 본발명의일양태는，
[6] (1)목적단백질을암호화하는발현백터로대장균을형질전환하는단계;
[7] (2)상기형질전환된대장균을배양배지에서배양하는단계； [8] (3)상기배양된대장균을포도당최소배지에접종하는단계;
[9] (4)상기최소배지에접종한대장균을배양하는단계；
[1이 (5)상기배양된대장균에안정동위원소로표지된아미노산을포함하는
아미노산흔합용액을투입하고，동위원소표지단백질의발현을유도하는단계;
[11] (6)상기아미노산용액이투입된대장균배양액을 15내지 25 °C의온도에서 8 내지 18시간동안배양하는단계；
[12] 를포함하는안정동위원소표지단백질의생산방법에관한것이다.
[13] 본발명에서상기단계 (1)의목적단백질은제한되지는않지만，
인간성장호르몬 (human growth hormone, hGH)일수있다.상기단계 (1)의 대장균은제한되지는않지만대장균 BL21균주를사용할수있으며， 37 °C에서 OD600값이 0.4내지으8에이를때까지배양할수있다.또한배양배지는 제한되지는않지만， 40내지 60 mg/mL카나마이신을포함하는 5내지 15 g/L 박토트립톤 (Bacto Tryptone), 3내지 8 g/L효모추출물 (yeast extract)및 5내지 15 g/L염화나트륨이녹아있는신선한 LB(Luria-Breta i)배지를사용할수있다. 또한본발명에서 ,상기단계 (5)의안정동위원소는제한되지는않지만, "C6^N4 -아르기닌 (arginine, Arg)일수있다.
[14] 본발명의다른양태로는,
[15] (1)목적단백질을암호화하는발현백터로대장균을형질전환하는단계;
[16] (2)상기형질전환된대장균을배양배지에서배양하는단계；
[17] (3)상기배양된대장균을포도당최소배지에접종하는단계；
[18] (4)상기최소배지에접종한대장균을배양하는단계；
[19] (5)상기배양된대장균에안정동위원소로표지된아미노산용액을투입하고, 동위원소표지단백질의발현을유도하는단계;
[20] (6)상기아미노산용액이투입된대장균배양액을 15내지 25 °C의온도에서 8 내지 18시간동안배양하는단계 ;
[21] (7)상기단계 (6)의대장균을완층용해용액으로용해하는단계;
[22] (8)상기대장균을초음파처리하고,원심분리하여동위원소표지단백질을
회수하는단계;및
[23] (9)상기안정동위원소표지단백질을정제하는단계；
[24] 를포함하는안정동위원소표지단백질의정제방법에관한것이다.
[25] 본발명에서상기단계 (1)의목적단백질은제한되지는않지만，
인간성장호르몬일수있으며，바람직하게는히스티딘표지된인간성장호르몬일 수있다.또한상기안정동위원소표지된아미노산은제한되지는않지만 i3C6 15N4
-아르기닌일수있다.
[26] 본발명에서，상기안정동위원소표지단백질의정제는제한되지는않지만
친화성크로마토그래피및음이온 -교환크로마토그래피중하나이상을이용할 수있으며,바람직하게상기친화성크로마토그래피의컬럼은 Ni-NTA컬럼을， 상기음이온-교환크로마토그래피의컬럼은모노큐 (Mono Q)컬럼을이용할수 있다.
[27]
[28] 본발명에서상기단계 (7)의용해완충용액은제한되지는않지만 0.3내지 0.8 mM EDTA, 0.5내지 L5 mg/mL라이소자임 (lysozyme), lx단백질분해효소 저해제흔합물 (protease inhibitor cocktail)및비이온성변성제 (detergent)를 포함하는 pH 7.5내지 8.5의 Tris-HCl일수있다.또한,상기비이온성변성제는 바람직하게는 0.01내지 2% (v/v)의트리톤 (Triton) X-100또는트원 (Tween) 20일 수있다.
[29] 또한본발명어ᅵ서,상기단계 (7)의용해완층용액은제한되지는않지만염이 포함되지않을수있으며，바람직하게는상기염은염화나트륨또는염화칼륨일 수있다.
[30] 본발명에서상기용해완충용액의사용량은제한되지는않지만，
대장균세포 (펠렛 ) 30 mg에대하여 0.25내지 2 mL일수있다.
[31]
[32] *본발명의또다른양태는，
[33] (1)목적단백질을암호화하는발현백터로대장균을형질전환하는단계;
[34] (2)상기형질전환된대장균을배¾배지에서배양하는단계;
[35] (3)상기배양된대장균을포도당최소배지에접종하는단계；
[36] (4)상기최소배지에접종한대장균을배양하는단계；
[37] (5)상기배양된대장균에안정동위원소로표지된아미노산용액을투입하고， 동위원소표지단백질의발현을유도하는단계;
[38] (6)상기아미노산용액이투입된대장균배양액을 15내지 25 °C의온도에서 8 내지 18시간동안배양하는단계;
[39] (7)상기단계 (6)의대장균을완충용해용액으로용해하는단계;
[40] (8)상기대장균을초음파처리하고，원심분리하여동위원소표지단백질을 회수하는단계;
[41] (9)상기안정동위원소표지단백질을정제하는단계；및
[42] (10)정제한안정동위원소표지단백질을 MALDI-TOF MS또는 HPLC로
분석하는단계;
[43] 를포함하는단백질분석방법에관한것이다.본발명에서상기 MALDI-TOF MS는제한되지는않지만， m/z 600내지 3,500의범위일수있으며，상기 HPLC의 컬럼의온도는제한되지는않지만 20내지 50 °C,유속은 αΐ내지 1.0 mL/min일 수있다.
발명의효과
[44] 본발명은대장균시스템에서안정동위원소표지 hGH을생산하기위해, 목적단백질을암호화하는발현백터를이용하여대장균을형질전환한후포도당 최소배지에접종하여배양하는과정에서안정동위원소표지된특정아미노산을 투입하여동위원소표지 hGH의대량발현을유도하고，이를추출과정에서 hGH 용해도를최대화하고，이를매우높은순도로정제하는방법및동위원소표지 hGH의분석방법에관한것이다.본발명을통해단백질의정량분석을위한 내부표준물질로이용할수있는안정동위원소표지단백질을높은효율로대량 발현하고，단백질추출과정에서용해도를극대화하여높은수득률을얻을수 있다.이후친화성크로마토그래피와이온-교환크로마토그래피를통해매우 높은순도의 hGH를정제할수있었으며,이를바탕으로다량의고순도
안정동위원소표지 SI-His-hGH를생산하였고, MALDI-TOF MS를이용하여 동위원소표지여부를성공적으로확인하였다.
도면의간단한설명
[45] 도 1은대장균에서재조합 hGH와 His-hGH의발현을나타낸것이다.
[46] M:단백질마커， U:형질전환후，발현유도되지않은세포, I：형질전환후， 발현유도된세포， Empty:형질전환되진않은세포， hGH:비태그 -hGH발현세포， His-hGH:히스티딘표지 -hGH발현세포.
[47] 도 2는최적화되지않은네이티브 (native)조건에서 His-hGH의정제를나타낸 것이다.
[48] L:세포용해물， S:상등액 (가용성단백질)， P:펠렛 (불용성단백질), El: elution
1, E2: elution 2, E3: elution 3， FT: flow through.
[49] 도 3은최적화된네이티브조건에서 His-hGH의정제를나타낸것이다.
[50] 1:발현유도되지않은세포， 2:발현유도된세포， 3:세포용해물, 4:펠렛 (불용성 단백질), 5:상등액 (가용성단백질)， 6: elution 1， 7: elution 2， 8: flow through, 9: 2차 elution 1, 10: 2차 elution 2, 11: 2차 flow through.
[51] 도 4는대장균에서 His-hGH과 SI(stable isotope,안정동위원소표지 )-His-hGH의 발현을비교하여나타낸것이다.
[52] 도 5는최적화된네이티브조건에서 SI-His-hGH의 1차정제를나타낸것이다.
[53] 1:유도되지않은세포， 2:유도된세포， 3:세포용해물 , 4:팰렛 (불용성단백질),
5:상등액 (가용성단백질)， 6: elution 1, 7: elution 2， 8: elution 3, 9: flow through, 10: 비교대조군 LG-hGH (2 g), 11:비교대조군 LG-hGH (10 g).
[54] 도 6은 His-hGH를트립신처리한후예상되는펩타이드를나타낸것이다.
[55] 도 7은양성대조군 LG-hGH과 SI-His-hGH를트립신처리후발생하는 4종의 대표펩타이드들을 MALDI-TOF질량분석기를이용하여질량측정값을확인한 그림이다.
[56] 도 8은 m/z 1205.574의 LG-hGH트립신분해펩타이드의 Lift TOF TOF (MS/MS) 스펙트럼과 m/z 1215.429의 SI-His-hGH펩타이드의스펙트럼을비교하는 그림이다.
[57] 도 9는모노큐컬럼을사용한 His-hGH의이온-교환크로마토그래피를나타낸 것이다. [58] 도 10은모노큐분획의 silver staining분석결과를나타낸것이다.
[59] 도 11은 LG-hGH와정제된 His-hGH의 HPLC분석결과를나타낸것이다. 발명의실시를위한최선의형태
[60] 이하본발명을실시예와첨부된도면을참조하여상세히설명한다.그러나 이들은본발명을보다상세하게설명하기위한것으로，본발명의권리범위가 하기의실시예에의해한정되는것은아니다.
[61]
[62] (실시예 1)인간성장호르몬발현을위한백터의제조
[63] 재조합인간성장호르몬발현을위한인간성장호르몬유전자 (NCBI Reference:
NM_000515.3: 141-719)는 (주)바이오니아 (BIONEER, Daejeon, Korea)에합성을 의뢰하였고， 5'말단에 Nhel제한효소절단자리 (GCTAGC)와 3'말단에 Xhol 제한효소절단자리 (CTCGAG)를삽입하였다 (서열번호 1).상기합성된 cDNA를 제한효소 Nhel와 Xh이를이용하여 pET-28a(Novagen, Madison, WI, USA)에 ligation시켜 N-말단에 6-히스티딘표지와트름빈절단부위를갖는재조합 인간성장호르몬을발현하는 His-hGH발현백터를완성하였다.
[64] 또한，히스티딘표지가없는인간성장호르몬을발현하는 untagged hGH발현 백터의경우，정방향프라이머 5' -££ ggcgatgttcccaaccatt-3' (서열번호 2)와역방향 프라이머 5'-£i gagctagaagccacagct-3' (서열번호 3)을이용하여상기합성된 cDNA를주형으로하여중합효소연쇄반웅하여 5'말단에 Ncol제한효소 절단자리 (CCATGG)와 3'말단에 Xhol제한효소절단자리 (CTCGAG)를포함하는 cDNA를제작하였다.이증합효소연쇄반웅의결과물은 Nc이과 Xh이의 제한부위로잘리고,표지가없는재조합인간성장호르몬 (untagged hGH)을 발현하기위한 pET28a발현백터의 Nc이과 Xhol제한부위들과이어졌다.
발현하고자하는유전자의뉴클레오티드서열은자동순서화를통하여 확인되었다.
[65]
[66] (실시예 2)대장균발현시스템을이용한인간성장호르몬발현및추출
[67] His-hGH발현재조합 DNA 3종을대장균 BL21(DE3)에형질전환하였다. 50 mg/mL카나마이신을포함하는 250 mL플라스크에상기대장균을접종하고 37 °C에서하룻밤배양하였다.배양액 5 mL을 10,000 x g에서 5분간원심분리한후 0.2중량 %포도당을함유한 M9배지 (Cat No. M8003; Technova Co. USA)로 세척하였다.처리한배양액을카나마이신을포함한 M9배지 500mL가담긴 4L 플라스크에접종하고 OD600약 0.6의값을가질때까지 37 °C에서배양하였다. 아미노산용액을만들기위해 200 mL증류수에아르기닌 (Arg)을제외한 19가지 아미노산 lg각각을넣어 25 X아미노산흔합물을제조한후 1 mL증류수에 방사성동위원소로표지된 "C15N-아르기닌 (Arg) 250 g을포함하는용액을 제조하였다. 25 X아미노산흔합물 20 mL과 250 mg/mL농도의 13C15 N-아르기닌 (Arg) 400 을넣고 30분간배양하였다.상기용액 ().5 mL는
SDS-PAGE수행을위해별도로보관하고,상기대장균배양액에서
인간성장호르몬의발현을유도하기위하여 0.5 M
베타-디 -1-갈락토피라노사이드 (β-D-l-thiogalactopyranoside, IPTG) 1 mL을넣어 단백질을발현시켰다.상기단백질을 SI-His-hGH라명명하였다. 16 °C에서 16 시간배양한후마찬가지로 0.5 mL를 SDS-PAGE수행을위해별도로보관하고， 나머지용액을 4 °C에서 10,000 x g로 20분간원심분리하여세포를수득한후 냉동하여보관하였다.회수한대장균세포를 25 mL용해완층용액 (1 mg/mL 라이소자임, 1 X프로테아제저해제칵테일 (로쉬，스페인)및 0.5 mM EDTA를 포함하는 50 mM Tris-HCl)과초음파를이용하여파쇄한후， 10,000 x g에서 20분 동안원심분리하여불용성 (pellete)및가용성 (soluble)분획을수득하여
SDS-PAGE를실시하였고코마쉬블루염색시약으로염색하였다.또한, 인간성장호르몬의분석을위하여이미지퀀트™TL 5.2분석
소프트웨어 (ImageQuant™ TL 5.2 analysis software)를사용하여
농도계 (densitometry)분석법으로가용성 (S)및불용성 (P)단백질양을
분석하였다.
[68] 도 1에서, His-hGH는단백질합성을유도하는 IPTG에의해발현되는것을 확인할수있다.상기결과로부터 His-hGH발현 DNA를이후실시예의대상으로 선정하였다.
[69]
[7이 (실시예 3)불용성분획의가용화를위한용해완층용액의최적조건
[71]
[72] 불용성분획을가용화하여가용성분획에포함할수있도록하는
용해완층용액의최적조성을확인하였다.상기실시예 2와동일한방법을 수행하여수득한불용성분획은하기표 1조성의용해완충용액을이용하여 최적의조성을확인하였다.
[73]
[74] 표 1
[Table 1]
불용성분획에서가용화단백질의수득을위한용해완층용액의조성
Figure imgf000008_0001
[76] (실시예 4)재조합단백질，인간성장호르몬의정제
[77] 상기실시예 1내지실시예 3에서확립한최적의인간성장호르몬발현및추출 조건을활용하여인간성장호르몬의추출후정제하였다.재조합
인간성장호르몬 (His-hGH및 SI-ffis-hGH)을발현시키는 E. coli BL21(DE3)은 250 mL배양배지에서배양하였고 16 °C에서 16시간동안유도되어수확하였다. 상기수확한세포를 25 mL용해완층용액 (0.5 mM EDTA, 0.1%트리톤 X-100, 1 mg/mL라이소자임및 1 X프로테아제저해제칵테일 (로쉬，스페인)을포함하는 pH 8.0 50 mM Tris-HCl)에녹여초음파처리한후 10,000 x g로 20분동안 원심분리한후수득하여정제하였다.
[78] (1) His-hGH및 SI-His-hGH의정제
[79] Ni-NTA컴럼욤사용하 ^화성크로마토그래피 (ᅳ affmitv chromatographv)로 1차 정제
[80] 원심분리후최적으로용해된 His-hGH및 SI-His-hGH상층액을 Ni-NTA
아가로스비즈 hnL가있는컬럼 (Qiagen, Valencia, CA, USA)에각각주입하였다. 컬럼에주입된 His-hGH및 SI-His-hGH단백질을컬럼부피의 3배되는표 2의 세척완충용액으로씻고，표 2의 10 mL용리완충용액 (elution buffer)으로 용리하여 His-hGH및 SI-His-hGH를 1차정제하였다.상기 His-hGH및
SI-His-hGH가포함된분획을각각음이온교환컬럼완층용액 (pH 8.0 50 mM Tris-HCl및 10%글리세를)으로투석하였다. 표 2
[Table 2]
친화성크로마토그래피를위한완층용액의조성
Figure imgf000009_0001
[83] *세척및용리완층용액으로상기조성을포함하는 pH 7.4의
be인산염완충용액 (Phosphate buffered saline; PBS)을사용하였다.
[84]
[85] 모노큐 (Mono 0)컴럼음사용하음이온 -교환크로마토그래피 (anion-exchange chromatography)로 2차정제
[86] 투석한분획들은음이온 -교환크로마토그래피인 5/50모노큐컬럼 (GE
Healthcare, USA)으로 0내지 500 mM염화나트륨농도구배에따른직선 변화도를주며용리하여 2차정제하였다.
[87] 도 2의레인 S(supematant, soluble protein)와레인 P(pellet, insoluble protein)를 보면，대부분의 His-hGH가불용성상태인것을확인할수있었다.따라서, Ni-NTA컬럼크로마토그래피를통해 E1-E3과같이오직소량의 His-hGH만을 회수할수있었다.상기실시예를통해최적화된조건에서단백질발현과 Ni-NTA친화성크로마토그래피를수행한결과는도 3과같다.레인 4와레인 5를 통해대부분의 His-hGH가가용성분획 (soluble fraction)에존재한다는것을 확인하였고,레인 6-7및 9-10에서이전실험결과의전체단백질수율인 2 mg/250mL배양액보다 15배이상높은 30 mg/250mL배양액을얻을수있음을 확인하였다.
[88] 도 4는 SI-His-hGH의발현여부를나타낸것으로,레인 2와레인 4를비교해 보면， I3C15N-Arg표지단백질과비표지단백질의젤이동성 (gel mobility)차이를 확인할수있었다.상기결과를바탕으로 Ni-NTA친화성컬럼크로마토그래피를 수행한결과는도 5와같다.레인 4와 5에서단백질의용해성을확인하였고，레인 6-8에서높은농도로분리된단백질을확인할수있었다.
[89]
[90] (실시예 5)정제된단백질의분석
[91] (1) MALDI-TOF질량분석기를이용한정제단백질분석
[92] MALDI-TOF질량분석기를이용한정제된단백질의분석을위해상기
실시예에서수득한정제된단백질을쿠마시스테인드젤 (Coomassie stained gel)에서표적단백질을분리하여트립신 (trypsin)을처리하는인-젤 다이제스트 (in-gel digest)방법을사용하여 MALDI-TOF MS를위한전처리를 수행하였다. His-hGH및 SI-His-hGH를포함하는젤조각을각각잘라낸후에 환원과알킬화를유도하고， 66 °C에서 10분동안배양한다.트립신은 22.5 ng^L 농도로 25 mM암모늄바이카보네이트 (ammonium bicarbonate, NH4HC03)에 녹여서젤조각과 37 °C에서밤새반웅시켜펩타이드형성을유도하였다.이렇게 주줄된팹타이드를 MALDI-TOF(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry)로분석하였다.상기수득한단백질 1 mg/mL을 MALDI메트릭스 (matrix)인
알파-시아노 _4-하이드톡시나믹산 (a-cyano-4-hydroxycinnamic acid)과 1: 1 (v/v)의 비율로흔합하여 MALDI질량분석플레이트에점적 (spoting)한다음,오토플렉스 III스마트빔 (Autoflex III Smartbeam,브루커달토닉스사，미국)장치에서 분석하였다.외부부족인펩티드및단백질보정키트 (Sigma-Aldrich, USA)를 사용하였으며，질량분석스펙트럼은 600내지 3,500 m/z범위의양이온모드에서 수행하여 His-hGH및 SI-His-hGH의질량을확인하였다.
[93]
[94] (2) HPLC를이용한정제된단백질의순도확인
[95] 상기음이은-교환크로마토그래피로확인된 His-hGH분획을프로미넌스
UFLC시스템 (Prominence UFLC system, Shimadzu)과 Kinetex CI 8컬럼 (2.6 μπι, 50 χ 2.10 mm; Phenomenex, Torrance, CA, USA)으로분석하였다.양성대조군으로 LG-hGH(LG생명과학，대한민국)을사용하였다.완층용액은 A(0.1%
Trifluoroacatic acid(TFA) in water)및 B(0.1% Trifluoroacatic acid(TFA) in acetonitrile(ACN))를사용하였으며，용리완층용액 B의직선변화도는 7분간 35%에서 100%로직선기울기를주어 40 oC에서용리하였다.유속은 0.4 mL/min이었으며 , 220 nm파장에서 UV흡광도를측정하였다.
[96]
[97] 도 6은 His-hGH을트립신으로처리했올때형성되는예상펩타이드들의
질량값을나타낸것이다.도 7은비교대조군 LG-hGH과 SI-His-hGH를트립신 처리후발생하는펩타이드들을 MALDI-TOF질량분석기를이용하여
질량측정값을확인한결과로써, SI-His-hGH분석한 4종의펩타이드들이 C6 15N4 -아르기닌으로인해 m/z값이약 10정도차이가발생함을확인하였고이는 SI-His-hGH의합성이성공적임을나타낸다.이중비교대조군의 m/z 1205.5에서 검출된 parent이은과 SI-His-hGH의 m/z 1215.4에서검출된 parent이온을
MALDI-TOF/TOF (MS/MS)를위한 LIFT모드와 FlexControl(Bruker,독일)을 이용하여추가적으로분석한결과는도 8과같다.도 8의스펙트럼에서여러 daughter이온의형성을확인하였고,이증 13C6 15N4ᅵ아르기닌을포함하는모든 이온들이 m/z값에서 10정도차이가발생함을확인하여다.또한 Mascot 소프트웨어 (Matrix Science Inc,영국)에서분석한결과예상대로 hGH의특정 • 펩타이드임을확인하였다.
[98]
[99] 도 9에서 SI-His-hGH의 2차정제를위해이온 -교환크로마토그래피를실시한 후결과를나타낸것으로,용출시간 15분경에검출되는 SI-His-hGH의분획을 분리하였다.도 10에서는 Silver staining을통해상기분획에서순수하게정제된 SI-His-hGH를확인하였고，다른불순물과잘분리되었음을확인하였다.도 10에서확인된 SI-His-hGH의분획을 HPLC로분석한결과를도 11에도시하였다. 양성대조군인 LG-hGH과 SI-His-hGH를분석한결과，정제한 SI-His-hGH이높은 수준의순도를가지고있음을확인하였다.
서열목록 Free Text
[100] 서열목록은서열목록파일로첨부하였다.

Claims

청구범위
(1)목적단백질을암호화하는발현백터로대장균을
형질전환하는단계;
(2)상기형질전환된대장균을배양배지에서배양하는단계;
(3)상기배양된대장균을포도당최소배지에접종하는단계;
(4)상기최소배지에접종한대장균을배양하는단계;
(5)상기배양된대장균에안정동위원소로표지된아미노산 용액을투입하고，동위원소표지단백질의발현을유도하는단계;
(6)상기아미노산용액이투입된대장균배양액을 15내지 25 °C의 온도에서 8내지 18시간동안배양하는단계;
를포함하는안정동위원소표지단백질의생산방법.
제 1항에있어서，
상기단계 (1)의목적단백질은인간성장호르몬인안정동위원소 표지단백질의생산방법.
제 1항에있어서，
상기단계 (5)의안정동위원소는 13C N-아르기닌인안정동위원소 표지단백질의생산방법 .
(1)목적단백질을암호화하는발현백터로대장균을
형질전환하는단계;
(2)상기형질전환된대장균을배양배지에서배양하는단계;
(3)상기배양된대장균을포도당최소배지에접종하는단계;
(4)상기최소배지에접종한대장균을배양하는단계;
(5)상기배양된대장균에안정동위원소로표지된아미노산 용액을투입하고，동위원소표지단백질의발현을유도하는단계;
(6)상기아미노산용액이투입된대장균배양액을 15내지 25 0C의 온도에서 8내지 18시간동안배양하는단계;
(7)상기단계 (6)의대장균을완충용해용액으로용해하는단계;
(8)상기대장균을초음파처리하고,원심분리하여동위원소표지 단백질을회수하는단계;및
(9)상기안정동위원소표지단백질을정제하는단계;
를포함하는안정동위원소표지단백질의정제방법 .
제 4항에있어서，
상기단계 (1)의목적단백질은인간성장호르몬인안정동위원소 표지단백질의정제방법.
제 5항에있어서，
상기목적단백질은히스티딘표지된인간성장호르몬인 안정동위원소표지단백질의정제방법. [청구항 7] 제 4항에있어서，
상기안정동위원소표지된아미노산은 13C15N-아르기닌인 안정동위원소표지단백질의정제방법.
[청구항 8] 제 4항에있어서，
상기안정동위원소표지단백질의정제는친화성크로마토그래피 및음이온 -교환크로마토그래피중하나이상을이용하는 안정동위원소표지단백질의정제방법 .
[청구항 9] 제 8항에있어서，
상기친화성크로마토그래피의컬럼은 Ni-NTA컬럼인
안정동위원소표지단백질의정제방법ᅳ
[청구항 10] 제 8항에있어서,
상기음이온-교환크로마토그래피의컬럼은모노큐컬럼인 안정동위원소표지단백질의정제방법.
[청구항 11] 제 4항에있어서,
상기단계 (9)의안정동위원소표지단백질은생물학적활성이 저해되지않는안정동위원소표지단백질의정제방법ᅳ
[청구항 12] 제 4항에있어서,
상기단계 (7)의용해완충용액은비이온성변성제를포함하는 안정동위원소표지단백질의정제방법 .
[청구항 13] 제 12항에있어서,
상기비이온성변성제는 0.01내지 2% (v/v)의트리톤 X-100또는 트원 20인안정동위원소표지단백질의정제방법.
[청구항 14] 제 4항에있어서，
상기단계 (7)의용해완충용액은염이포함되지않는
안정동위원소표지단백질의정제방법.
[청구항 15] 제 M항에있어서，
상기염은염화나트륨또는염화칼륨인안정동위원소표지 단백질의정제방법.
[청구항 16] 제 4항에있어서，
상기용해완층용액의사용량은대장균세포 (펠렛) 30 mg에대하여 0.25내지 2 mL인안정동위원소표지단백질의정제방법.
[청구항 Π] (1)목적단백질을암호화하는발현백터로대장균을
형질전환하는단계;
(2)상기형질전환된대장균을배양배지에서배양하는단계;
(3)상기배양된대장균을포도당최소배지에접종하는단계；
(4)상기최소배지에접종한대장균을배양하는단계;
(5)상기배양된대장균에안정동위원소로표지된아미노산 용액을투입하고，동위원소표지단백질의발현을유도하는단계; (6)상기아미노산용액이투입된대장균배양액을 15내지 25 0C의 온도에서 8내지 18시간동안배양하는단계;
(7)상기단계 (6)의대장균을완층용해용액으로용해하는단계;
(8)상기대장균을초음파처리하고,원심분리하여동위원소표지 단백질을회수하는단계;
(9)상기안정동위원소표지단백질을정제하는단계;및
(10)정제한안정동위원소표지단백질을 MALDI-TOF MS또는 HPLC로분석하는단계;
를포함하는안정동위원소표지단백질의분석방법.
[청구항 18] 제 Π항에있어서，
상기 MALDI-TOF MS는 m/z 600내지 3,500의범위에서질량 스펙트럼을확인하는안정동위원소표지단백질의분석방법.
[청구항 I9] 제 Π항에있어서,
상기 HPLC의컬럼의온도는 20내지 50 °C,유속은 0.1내지 1.0 mLAnin으로설정하여검출하는안정동위원소표지단백질의분석 방법.
PCT/KR2015/001063 2014-02-19 2015-02-02 안정동위원소 표지 단백질 생산, 정제 및 분석 방법 Ceased WO2015126077A1 (ko)

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