KR20200119743A - The mass production process of teriparatide using E.coli - Google Patents
The mass production process of teriparatide using E.coli Download PDFInfo
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- KR20200119743A KR20200119743A KR1020200043975A KR20200043975A KR20200119743A KR 20200119743 A KR20200119743 A KR 20200119743A KR 1020200043975 A KR1020200043975 A KR 1020200043975A KR 20200043975 A KR20200043975 A KR 20200043975A KR 20200119743 A KR20200119743 A KR 20200119743A
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- teriparatide
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Abstract
Description
본 발명은 대장균을 이용한 테리파라타이드(teriparatide)의 대량생산공정에 관한 것으로, 보다 구체적으로 본 발명은 테리파라타이드 융합단백질을 발현시킨 후, 흡착 크로마토그래피에 의한 1차 정제단계; 및 TEV(Tobacco etch virus) 단백질분해효소를 처리하는 단계를 포함하는, 테리파라타이드 대량 생산방법에 관한 것이다. The present invention relates to a mass production process of teriparatide using E. coli, and more specifically, the present invention relates to a first purification step by adsorption chromatography after expressing the teriparatide fusion protein; And it relates to a method for mass production of teriparatide comprising the step of treating a TEV (Tobacco etch virus) protease.
또한, 본 발명은 서열번호 1의 염기서열에 의해 코딩되는 테리파라타이드 융합단백질; 상기 서열을 포함하는 테리파라타이드 융합단백질 발현벡터; 및 상기 발현 벡터가 도입된 형질전환체에 관한 것이다. In addition, the present invention is a teriparatide fusion protein encoded by the nucleotide sequence of SEQ ID NO: 1; Teriparatide fusion protein expression vector comprising the sequence; And to a transformant into which the expression vector has been introduced.
부갑상선 호르몬(PTH)은 포유동물의 부갑상선으로부터 분비되는 84개 아미노산으로 이루어진 물질로 뼈를 포함한 다양한 조직에서 혈청 칼슘 농도를 제어하는 기능을 가진다. 사람의 일부 PTH 형태에 관한 연구에서 뼈에서의 PTH의 동화 효과가 나타났으며, 이는 골다공증 및 이와 관련된 뼈 질환의 치료를 위한 PTH의 용도에 큰 관심을 불러 일으켰다.Parathyroid hormone (PTH) is a substance consisting of 84 amino acids secreted from the parathyroid glands of mammals and has a function of controlling serum calcium concentration in various tissues including bones. Studies on some forms of PTH in humans have shown an anabolic effect of PTH in bone, which has aroused great interest in the use of PTH for the treatment of osteoporosis and related bone diseases.
예를 들어, 모든 발표된 보고에서 전체 길이의 호르몬과 생물학적 동등성을 가진다고 간주되는 소 및 사람의 호르몬의 N-말단의 34개 아미노산을 사용하여, 사람에 있어서 부갑상선 호르몬이 특히 피하 및 정맥내 경로에 의해 박동성 양식으로 투여될 때 뼈의 성장을 촉진시키는 것으로 나타났다. PTH의 약간 상이한 형태인 사람의 PTH(1-38)도 이와 유사한 결과를 나타낸다. rhPTH(1-34)라고도 불리는 테리파라타이드는 유전자 재조합 부갑상선 호르몬에 해당되는 물질로, 정제된 결정 형태의 테리파라타이드의 합성과 절편화된 PTH의 제조 및 정제 방법을 개시하고 있는 US6590081B1을 비롯하여 그 제조방법에 대하여 개시하고 있는 문헌은 존재하나, 본 발명과 같은 흡착크로마토그래피 및 TEV 단백질 분해효소를 사용한 테리파라타이드 생산방법에 관해서는 알려져 있지 않다. For example, using 34 amino acids at the N-terminus of bovine and human hormones, which are considered bioequivalent to full-length hormones in all published reports, parathyroid hormones in humans, especially in subcutaneous and intravenous pathways, are used. It has been shown to promote bone growth when administered in a pulsatile mode. Human PTH (1-38), a slightly different form of PTH, has similar results. Teriparatide, also called rhPTH(1-34), is a substance corresponding to genetically modified parathyroid hormone, including US6590081B1, which discloses the synthesis of purified crystalline teriparatide and the production and purification method of fragmented PTH. Although there are documents that disclose the preparation method, the method for producing teriparatide using the same adsorption chromatography and TEV protease as in the present invention is not known.
따라서, 본 발명자는 흡착크로마토그래피 및 TEV 단백질 분해효소를 사용하는 본 발명의 생산방법에 의할 경우, 고순도의 테리파라타이드를 경제적으로 대량생산할 수 있다는 장점이 있다는 점에 착안하여 본 발명을 완성하였다. Therefore, the present inventors completed the present invention by focusing on the advantage of economically mass-producing high-purity teriparatide by the production method of the present invention using adsorption chromatography and TEV protease. .
본 발명의 하나의 목적은 테리파라타이드(teriparatide) 융합단백질, 흡착 크로마토그래피 및 TEV(Tobacco etch virus) 단백질분해효소를 이용한 테리파라타이드의 대량 생산방법을 제공하는 것이다. One object of the present invention is to provide a method for mass production of teriparatide using teriparatide fusion protein, adsorption chromatography, and TEV (Tobacco etch virus) protease.
본 발명의 다른 하나의 목적은 서열번호 1의 염기서열에 의해 코딩되는 테리파라타이드 융합단백질; 상기 서열번호 1의 염기서열을 포함하는 테리파라타이드 융합단백질 발현벡터; 및 상기 발현벡터가 도입된 형질전환체를 제공하는 것이다. Another object of the present invention is a teriparatide fusion protein encoded by the nucleotide sequence of SEQ ID NO: 1; Teriparatide fusion protein expression vector comprising the nucleotide sequence of SEQ ID NO: 1; And it is to provide a transformant into which the expression vector was introduced.
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 발명에서는 테리파라타이드(teriparatide) 융합단백질, 흡착 크로마토그래피 및 TEV(Tobacco etch virus) 단백질분해효소를 이용한 테리파라타이드의 생산방법을 통해 기존의 생산방법보다 경제적으로 고순도의 테리파라타이드를 생산할 수 있음을 확인할 수 있어 대량 생산방법에 적합함을 확인하였으며, 본 발명은 이에 기초한다. In the present invention, through the production method of teriparatide using teriparatide fusion protein, adsorption chromatography, and TEV (Tobacco etch virus) protease, it is possible to produce teriparatide of high purity more economically than the conventional production method. It was confirmed that it was confirmed that it was suitable for mass production, and the present invention is based on this.
이하 본 발명의 구성을 상세히 설명한다. Hereinafter, the configuration of the present invention will be described in detail.
상기 목적을 달성하기 위한, 본 발명의 하나의 양태는 테리파라타이드를 대량생산하는 방법을 제공한다. In order to achieve the above object, one aspect of the present invention provides a method for mass-producing teriparatide.
구체적으로, 본 발명에서 제공하는 테리파라타이드의 대량 생산방법은, Specifically, the mass production method of teriparatide provided by the present invention,
(a) 테리파라타이드(teriparatide) 융합단백질을 코딩하는 폴리뉴클레오티드를 포함하는 발현벡터가 도입된 형질전환체를 배양하는 단계;(a) culturing a transformant into which an expression vector containing a polynucleotide encoding a teriparatide fusion protein has been introduced;
(b) 상기 배양된 형질전환체, 이의 배양물 또는 이의 파쇄물을 흡착 크로마토그래피에 적용하여 테리파라타이드 융합단백질을 분리하는 1차 정제단계;(b) a first purification step of separating the teriparatide fusion protein by applying the cultured transformant, its culture or its lysate to adsorption chromatography;
(c) 상기 제1차 정제단계에서 수득된 융합단백질에 TEV(Tobacco etch virus) 단백질분해효소를 처리하는 단계;(c) treating the fusion protein obtained in the first purification step with a TEV (Tobacco etch virus) protease;
(d) 상기 단백질분해효소 처리 후 절단된 테리파라타이드를 이온교환수지 컬럼 크로마토그래피를 이용하여 분리하는 2차 정제단계; 및(d) a second purification step of separating teriparatide cut after the protease treatment by using ion exchange resin column chromatography; And
(e) 상기 분리된 테리파라타이드를 역상 컬럼 크로마토그래피로 정제하는 3차 정제단계를 포함한다. (e) a third purification step of purifying the separated teriparatide by reverse phase column chromatography.
본 발명의 용어, "테리파라타이드(teriparatide) 융합단백질"이란, 유전자 재조합 부갑상선 호르몬(rhPTH(1-34))으로 불리는 테리파라타이드가 포함된 단백질로서, 본 발명의 일 구체예로서 서열번호 1에 의해 코딩 될 수 있다. 상기 서열번호 1은 TEV 단백질 분해효소 인식 부위가 포함될 수 있으며, 테리파라타이드 융합단백질의 정제 과정에서 상기 인식부위를 TEV 단백질분해효소가 인식하여 테리파라타이드만을 수득할 수 있다. The term "teriparatide fusion protein" of the present invention is a protein containing teriparatide called genetically recombinant parathyroid hormone (rhPTH(1-34)), and as a specific example of the present invention, SEQ ID NO: 1 Can be coded by The TEV protease recognition site may be included in SEQ ID NO: 1, and TEV protease recognition site may be recognized by TEV protease during purification of the teriparatide fusion protein to obtain only teriparatide.
본 발명의 상기 테리파라타이드 융합단백질은 이를 코딩하는 염기서열을 포함하는 발현벡터 및 상기 벡터가 도입된 형질전환체를 배양 및 정제하는 과정을 통해 수득될 수 있다. The teriparatide fusion protein of the present invention can be obtained through a process of culturing and purifying an expression vector including a nucleotide sequence encoding it and a transformant into which the vector has been introduced.
또한, 본 발명에서 제공하는 테리파라타이드를 대량생산방법의 (b) 단계는 (b1) 상기 배양된 형질전환체, 이의 배양물 또는 이의 파쇄물에서 봉입체 형태의 융합단백질을 수득하는 단계; (b2) 상기 수득한 융합단백질을 수세하는 단계; 및 (b3) 수세 후 균질화하는 단계에 의해 수행될 수 있다. In addition, step (b) of the mass production method of teriparatide provided by the present invention includes the steps of (b1) obtaining a fusion protein in the form of an inclusion body from the cultured transformant, its culture, or its lysate; (b2) washing the obtained fusion protein with water; And (b3) homogenizing after washing with water.
상기 형질전환체를 배양함에 따라 이의 배양물 또는 파쇄물에서 테리파라타이드 융합단백질의 수득이 가능하다. By culturing the transformant, it is possible to obtain a teriparatide fusion protein from its culture or lysate.
본 발명의 용어, "배양"이란, 미생물을 적당히 인공적으로 조절한 환경조건에서 생육시키는 방법을 의미한다. 본 발명에 있어서, 상기 형질전환체를 배양하는 방법은 당업계에 널리 알려져 있는 방법을 이용하여 수행할 수 있다. 구체적으로 상기 배양은 본 발명의 융합단백질을 발현시켜서 생산할 수 있는 한 특별히 이에 제한되지 않으나, 배치 공정 또는 주입 배치 또는 반복 주입 배치 공정(fed batch or repeated fed batch process)에서 연속식으로 배양할 수 있다.The term "culture" of the present invention means a method of growing microorganisms in an appropriately artificially controlled environmental condition. In the present invention, the method of culturing the transformant may be performed using a method widely known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing the fusion protein of the present invention, but can be continuously cultured in a batch process, an injection batch, or a fed batch or repeated fed batch process. .
일 구체예로, 고밀도 세포 배양(high cell density culture, HCDC)하기 위해, 유가식 배양(fed batch fermentation)을 할 수 있으나, 이에 제한 되는 것은 아니다. In one embodiment, for high cell density culture (HCDC), fed batch fermentation may be performed, but is not limited thereto.
배양에 사용되는 배지는 적당한 탄소원, 질소원, 아미노산, 비타민 등을 함유한 통상의 배지 내에서 호기성 조건 하에서 온도, pH 등을 조절하면서 적절한 방식으로 특정 균주의 요건을 충족해야 한다. 사용될 수 있는 탄소원으로는 글루코즈 및 자일로즈의 혼합당을 주 탄소원으로 사용하며 이외에 수크로즈, 락토즈, 프락토즈, 말토즈, 전분, 셀룰로즈와 같은 당 및 탄수화물, 대두유, 해바라기유, 피마자유, 코코넛유 등과 같은 오일 및 지방, 팔미트산, 스테아린산, 리놀레산과 같은 지방산, 글리세롤, 에탄올과 같은 알코올, 아세트산과 같은 유기산이 포함된다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있다. 사용될 수 있는 질소원으로는 암모니아, 황산암모늄, 염화암모늄, 초산암모늄, 인산암모늄, 탄산안모늄, 및 질산암모늄과 같은 무기질소원; 글루탐산, 메티오닌, 글루타민과 같은 아미노산 및 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 맥아 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크 또는 그의 분해생성물 등 유기질소원이 사용될 수 있다. 이들 질소원은 단독 또는 조합되어 사용될 수 있다. 상기 배지에는 인원으로서 인산 제1칼륨, 인산 제2칼륨 및 대응되는 소듐-함유 염이 포함될 수 있다. 사용될 수 있는 인원으로는 인산이수소칼륨 또는 인산수소이칼륨 또는 상응하는 나트륨-함유 염이 포함된다. 또한, 무기화합물로는 염화나트륨, 염화칼슘, 염화철, 황산마그네슘, 황산철, 황산망간 및 탄산칼슘 등이 사용될 수 있다. 마지막으로, 상기 물질에 더하여 아미노산 및 비타민과 같은 필수 성장 물질이 사용될 수 있다. The medium used for cultivation must meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc. As the carbon source that can be used, a mixed sugar of glucose and xylose is used as the main carbon source. In addition, sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose, soybean oil, sunflower oil, castor oil, coconut Oils and fats such as oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid are included. These materials can be used individually or as a mixture. Examples of nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products, etc. I can. These nitrogen sources may be used alone or in combination. The medium may contain first potassium phosphate, second potassium phosphate, and corresponding sodium-containing salt as personnel. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used as the inorganic compound. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the above substances.
또한, 배양 배지에 적절한 전구체들이 사용될 수 있다. 상기된 원료들은 배양과정에서 배양물에 적절한 방식에 의해 회분식, 유가식 또는 연속식으로 첨가될 수 있으나, 특별히 이에 제한되지는 않는다. 수산화나트륨, 수산화칼륨, 암모니아와 같은 기초 화합물 또는 인산 또는 황산과 같은 산 화합물을 적절한 방식으로 사용하여 배양물의 pH를 조절할 수 있다. In addition, precursors suitable for the culture medium may be used. The above-described raw materials may be added in a batch, fed-batch, or continuous manner to the culture during the culture process, but are not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.
이때, 배양 pH는 pH 6.5 내지 7.5가 유지되도록 배양할 수 있으며, 구체적으로 pH 6.8~7.0일 수 있고, 더욱 구체적으로 pH 6.8일 수 있으나, 이에 제한 되는 것은 아니다. In this case, the culture pH may be cultured to maintain a pH of 6.5 to 7.5, and specifically, may be a pH of 6.8 to 7.0, and more specifically, a pH of 6.8, but is not limited thereto.
또한, 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 호기 상태를 유지하기 위해 배양물 내로 산소 또는 산소-함유 기체(예, 공기)를 주입한다. In addition, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Oxygen or oxygen-containing gas (eg, air) is injected into the culture to maintain aerobic conditions.
배양물의 온도는 27℃내지 38℃일 수 있으나, 바람직하게는 35℃내지 37℃일 수 있다. 배양은 상기 테리파라타이드 융합단백질의 생성량이 최대로 얻어질 때까지 계속할 수 있다. 배양 시간은 10 내지 100 시간일 수 있으며, 구체적으로 20 내지 40시간일 수 있으나, 이에 제한되는 것은 아니다. The temperature of the culture may be 27°C to 38°C, but preferably 35°C to 37°C. The cultivation may be continued until the maximum amount of the teriparatide fusion protein is obtained. The incubation time may be 10 to 100 hours, and specifically, may be 20 to 40 hours, but is not limited thereto.
또한, 배양 산소조건은 25 내지 40%일 수 있으며, 구체적으로 30%일 수 있으나 이에 제한 되는 것은 아니다. In addition, the culture oxygen condition may be 25 to 40%, specifically 30%, but is not limited thereto.
본 발명의 일 실시예에서는, 형질전환체로 대장균을 이용하여 37℃의 온도, 30%의 산소조건,pH 6.8을 유지하도록 배양하였으며, 테리파라타이드의 발현 유도를 위하여 IPTG(Isopropyl β-thiogalactopyranoside)를 첨가하여 배양한 결과, 테리파라타이드 융합단백질이 발현됨을 확인할 수 있었다. In one embodiment of the present invention, E. coli was used as a transformant to maintain a temperature of 37°C, an oxygen condition of 30%, and a pH of 6.8, and IPTG (Isopropyl β-thiogalactopyranoside) was used to induce the expression of teriparatide. As a result of adding and culturing, it was confirmed that the teriparatide fusion protein was expressed.
이때, 상기 배양을 거친 형질전환체의 파쇄물은 특별히 제한되지는 않으나, 상기 형질전환체를 초음파 처리, 물리적인 분쇄, 급속동결 및 해동 등의 다양한 방법을 통해 수득할 수 있다. At this time, the lysate of the transformant that has undergone the culture is not particularly limited, but the transformant may be obtained through various methods such as ultrasonic treatment, physical pulverization, rapid freezing and thawing.
상기 형질전환체의 파쇄물로부터 수득한 융합단백질은 수세과정을 거칠 수 있으며, 일 구체예로 봉입체(Inclusion body)형태로 수득된 융합단백질을 수세할 수 있는 용액이면 특별히 제한이 없으며, 일 구체예로는 증류수 또는 완충액(washing buffer)을 사용할 수 있다. The fusion protein obtained from the lysate of the transformant may be washed with water, and as an example, there is no particular limitation as long as it is a solution capable of washing the fusion protein obtained in the form of an inclusion body with water. Distilled water or a washing buffer may be used.
상기의 방법에 의해 수득된 융합단백질은 흡착 컬럼 크로마토그래피를 이용하여 1차정제 과정을 거칠 수 있는데, 상기 흡착 컬럼 크로마토그래피를 사용함으로 인하여 기존에 단백질 정제에 사용하였던 His-tag를 이용한 친화 크로마토그래피를 대체할 수 있다. The fusion protein obtained by the above method can be subjected to a primary purification process using adsorption column chromatography.By using the adsorption column chromatography, affinity chromatography using the His-tag previously used for protein purification Can be substituted for
기존 친화 크로마토그래피는 단백질 정제에 사용되기는 하였으나, 비용적인 면에서 부담이 존재하여 대량생산화하는 데에 어려움이 있었다. 다만, 본 발명에서는 흡착 컬럼 크로마토그래피를 사용하여 기존에 사용하였던 친화 크로마토그래피와 동등 또는 그 이상의 정제효과를 보임을 확인함에 따라 이를 대체할 수 있는 정제법으로 대량생산에 적합한 방법임을 확인하였다(도4 내지 도6 참조). Conventional affinity chromatography was used for protein purification, but it was difficult to mass-produce due to a burden in terms of cost. However, in the present invention, it was confirmed that the purification effect equivalent to or higher than that of the previously used affinity chromatography was shown using adsorption column chromatography, and thus it was confirmed that it is a method suitable for mass production as a purification method that can replace it (Fig. 4 to 6).
본 발명에서 사용하는 흡착 컬럼 크로마토그래피는 그 종류에 특별히 제한이 있지는 않으나, HP20 흡착크로마토그래피 또는 GS20 흡착크로마토그래피를 사용할 수 있으며, 또는 이의 조합을 사용하여 1차 정제 단계를 수행할 수 있다. The adsorption column chromatography used in the present invention is not particularly limited in its kind, but HP20 adsorption chromatography or GS20 adsorption chromatography may be used, or a first purification step may be performed using a combination thereof.
또한, 본 발명에서 제공하는 테리파라타이드를 대량생산방법은 상기 흡착 크로마토그래피에 의해 융합단백질을 정제한 이후, 침전법을 추가로 수행할 수 있다. In addition, in the mass production method of teriparatide provided by the present invention, after the fusion protein is purified by the adsorption chromatography, a precipitation method may be additionally performed.
상기 침전법은 통상 사용될 수 있는 염을 이용하여 수행될 수 있으며, 염의 종류에 제한되는 것은 아니다. 침전법을 추가할 경우, 정제가 필요한 물질 자체의 볼륨을 줄일 수 있어 정제과정의 시간을 단축시킬 수 있는 장점이 있다. The precipitation method may be carried out using a salt that can be used in general, and is not limited to the type of salt. In the case of adding a precipitation method, the volume of the material that needs to be purified can be reduced, thereby reducing the time of the purification process.
또한, 본 발명에서 제공하는 테리파라타이드를 대량생산방법의 (c)단계에서 사용되는 단백질 분해효소는 상기 융합 단백질로부터 테리파라타이드를 분리할 수 있는 것을 사용할 수 있는데, 바람직하게는 TEV(tobacco etch virus) 단백질분해효소를 사용할 수 있다. In addition, the proteolytic enzyme used in step (c) of the mass production method of teriparatide provided in the present invention may be one capable of separating teriparatide from the fusion protein, preferably TEV (tobacco etch virus) protease can be used.
상기 (c) 단계의 TEV 단백질분해효소에 의한 처리는 TEV 단백질 분해효소의 활성에 최적인 조건에서 처리될 수 있으며, 특별히 조건이 제한되는 것은 아니다. The treatment with the TEV protease in step (c) may be performed under conditions optimal for the activity of the TEV protease, and the conditions are not particularly limited.
상기 TEV 단백질분해효소에 의한 처리는 TEV(tobacco etch virus) 단백질분해효소는 융합단백질과의 처리 양에 대한 비율이 1:1, 1:2, 1:4, 1:8, 1:12, 1:16, 1:32, 1:64, 1:128일 수 있으나, 이에 제한되는 것은 아니다. The treatment with the TEV protease has a ratio of TEV (tobacco etch virus) protease to the amount of treatment with the fusion protein: 1:1, 1:2, 1:4, 1:8, 1:12, 1 :16, 1:32, 1:64, 1:128 may be, but is not limited thereto.
다만, 상기 TEV(tobacco etch virus) 단백질분해효소는 융합단백질 대비 0.01 내지 0.1 중량%로 포함 될 수 있으며, 20 내지 40℃ 에서 3 내지 6 시간 동안 처리될 수 있다. 바람직하게는, 상기 TEV(tobacco etch virus) 단백질분해효소는 융합단백질 대비 0.06 내지 0.07 중량%로 포함하고, 30℃에서 4 내지 5 시간 동안 처리될 수 있다. 상기 조건에 의할 때, TEV(tobacco etch virus) 단백질분해효소에 의한 테리파라타이드의 절단 효과가 뛰어남을 확인할 수 있었다(도 8참조). However, the TEV (tobacco etch virus) protease may be contained in an amount of 0.01 to 0.1% by weight relative to the fusion protein, and may be treated at 20 to 40°C for 3 to 6 hours. Preferably, the TEV (tobacco etch virus) protease is contained in an amount of 0.06 to 0.07% by weight relative to the fusion protein, and may be treated at 30° C. for 4 to 5 hours. Under the above conditions, it was confirmed that the cleavage effect of teriparatide by TEV (tobacco etch virus) protease was excellent (see FIG. 8).
또한, 본 발명에서 제공하는 테리파라타이드를 대량생산방법의 (d)단계인 이온교환수지 컬럼 크로마토그래피를 통한 2차 정제 및 (e)단계인 역상 컬럼 크로마토그래피로 정제하는 3차 정제단계를 거쳐 (c)단계에서 융합단백질 및 테리파라타이드 등의 혼합물로부터 테리파라타이드를 순수분리 할 수 있다. In addition, teriparatide provided in the present invention undergoes a second purification through ion exchange resin column chromatography, which is step (d) of the mass production method, and a third purification step of purifying by reverse-phase column chromatography, which is step (e). In step (c), teriparatide can be purely separated from a mixture of fusion proteins and teriparatide.
상기 이온교환수지 컬럼 크로마토그래피 및 역상컬럼 크로마토그래피는 테리파라타이드를 분리할 수 있는 것이면 그 종류에 특별히 제한이 없다. 다만, 본 발명의 일 실시예에서 이온교환수지 컬럼으로 SP 양이온교환 컬럼(cation exchange column)을 사용하였으나 이에 제한되는 것은 아니다. The ion-exchange resin column chromatography and reverse-phase column chromatography are not particularly limited as long as teriparatide can be separated. However, although an SP cation exchange column was used as the ion exchange resin column in an embodiment of the present invention, it is not limited thereto.
상기 2차 및 3차 정제에 의하여, 순도가 높은 테리파라타이드를 분리할 수 있었으며(도 9 내지 도 11 참조), 본원발명의 상기 방법에 의할 경우 테리파라타이드의 대량 생산에 적합함을 확인할 수 있었다. By the second and third purification, it was possible to separate high-purity teriparatide (see FIGS. 9 to 11), and it was confirmed that it is suitable for mass production of teriparatide when the method of the present invention is used. Could
상기 목적을 달성하기 위한, 본 발명의 다른 하나의 양태는 서열번호 1의 염기서열에 의해 코딩되는 테리파라타이드 융합단백질; 상기 서열번호 1의 염기서열을 포함하는 테리파라타이드 융합단백질 발현벡터; 및 상기 발현벡터가 도입된 형질전환체를 제공한다. In order to achieve the above object, another aspect of the present invention is a teriparatide fusion protein encoded by the nucleotide sequence of SEQ ID NO: 1; Teriparatide fusion protein expression vector comprising the nucleotide sequence of SEQ ID NO: 1; And it provides a transformant into which the expression vector was introduced.
본 발명의 테리파라타이드 융합단백질은 서열번호 1의 염기서열에 의해 코딩될 수 있다. 이때, 상기 용어 "테리파라타이드 융합단백질"은 전술한 바와 같다. The teriparatide fusion protein of the present invention may be encoded by the nucleotide sequence of SEQ ID NO: 1. In this case, the term "teriparatide fusion protein" is as described above.
본 발명의 발현벡터는 상기 서열번호 1의 염기서열을 포함할 수 있다. The expression vector of the present invention may include the nucleotide sequence of SEQ ID NO: 1.
또한, 본 발명의 일 구체예로, 상기 발현벡터는 P2 프로모터, Lac 오퍼레이터 및 암피실린 저항유전자 중 어느 하나 이상을 추가로 포함할 수 있으나, 이에 제한 되는 것은 아니다. In addition, as an embodiment of the present invention, the expression vector may further include any one or more of a P2 promoter, a Lac operator, and an ampicillin resistance gene, but is not limited thereto.
본 발명의 용어, "발현벡터"란 목적하는 숙주세포에서 목적 펩타이드를 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 의미한다. 상기 발현벡터는 개시코돈, 종결코돈, 프로모터, 오퍼레이터 등의 발현조절 요소들을 포함하는데, 상기 개시 코돈 및 종결 코돈은 일반적으로 폴리펩타이드를 암호화하는 뉴클레오티드 서열의 일부로 간주되며, 유전자 제작물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. The term "expression vector" of the present invention refers to a recombinant vector capable of expressing a peptide of interest in a host cell of interest, and refers to a gene construct comprising essential regulatory elements operatively linked to express a gene insert. The expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator. The start codon and the stop codon are generally considered to be part of the nucleotide sequence encoding the polypeptide, and when the gene construct is administered, the individual It must exhibit an action in and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.
본 발명의 용어 "작동가능하게 연결(operably linked)"이란, 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질 또는 RNA를 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 상태를 의미한다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동가능하게 연결되어 코딩서열의 발현에 영향을 미칠 수 있다. 발현 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다.The term "operably linked" of the present invention means a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function. . For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the expression of the coding sequence. The operative linkage with the expression vector may be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage may use enzymes generally known in the art.
또한, 상기 발현벡터는 세포 배양액으로부터 단백질의 분리를 촉진하기 위하여 융합 폴리펩타이드의 배출을 위한 시그널 서열을 포함할 수 있다. 특이적인 개시 시그널은 또한 삽입된 핵산 서열의 효율적인 번역에 필요할 수도 있다. 이들 시그널은 ATG 개시코돈 및 인접한 서열들을 포함한다. 어떤 경우에는, ATG 개시 코돈을 포함할 수 있는 외인성 번역 조절 시그널이 제공되어야 한다. 이들 외인성 번역 조절 시그널들 및 개시 코돈들은 다양한 천연 및 합성 공급원일 수 있다. 발현 효율은 적당한 전사 또는 번역 강화 인자의 도입에 의하여 증가될 수 있다.In addition, the expression vector may include a signal sequence for release of the fusion polypeptide in order to facilitate the separation of the protein from the cell culture medium. Specific initiation signals may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal should be provided that may include the ATG initiation codon. These exogenous translational control signals and initiation codons can be of a variety of natural and synthetic sources. Expression efficiency can be increased by introduction of appropriate transcriptional or translation enhancing factors.
아울러, 상기 발현벡터는 융합단백질의 검출을 용이하게 하기 위하여, 임의로 엔도펩티아이제를 사용하여 제거할 수 있는 단백질 태그를 추가로 포함할 수 있다. In addition, the expression vector may additionally include a protein tag that can be removed using an endopeptiase, in order to facilitate detection of the fusion protein.
본 발명의 용어 "태그(tag)"란, 정량가능한 활성 또는 특성을 나타내는 분자를 의미하며, 플로오레세인과 같은 화학적 형광물질(fluoracer), 형광 단백질(GFP) 또는 관련 단백질과 같은 폴리펩타이드 형광물질을 포함한 형광분자일 수도 있고; Myc 태그, 플래그(Flag) 태그, 히스티딘 태그, 루신 태그, IgG 태그, 스트랩타비딘 태그 등의 에피톱 태그일 수도 있다. 특히, 에피톱 태그를 사용할 경우, 바람직하게는 6개 이상의 아미노산 잔기로 구성되고, 보다 바람직하게는 8개 내지 50개의 아미노산 잔기로 구성된 펩타이드 태그를 사용할 수 있다.The term "tag" of the present invention means a molecule that exhibits quantifiable activity or properties, and a polypeptide fluorescent substance such as a chemical fluorescent substance such as fluorescein, a fluorescent protein (GFP), or a related protein It may be a fluorescent molecule including; It may be an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, and a strapavidin tag. In particular, when using an epitope tag, preferably a peptide tag composed of 6 or more amino acid residues, more preferably 8 to 50 amino acid residues may be used.
본 발명에 있어서, 상기 발현벡터의 종류는 본 발명의 융합단백질을 생산할 수 있는 한, 특별히 이에 제한되지 않으나, 바람직하게는 플라스미드 DNA, 파아지 DNA 등이 될 수 있고, 보다 바람직하게는 상업적으로 개발된 플라스미드(pUC18, pBAD, pIDTSAMRT-AMP 등), 대장균 유래 플라스미드(pYG601BR322, pBR325, pUC118, pUC119 등), 바실러스 서브틸리스 유래 플라스미드(pUB110, pTP5 등), 효모-유래 플라스미드(YEp13, YEp24, YCp50 등), 파아지 DNA(Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP 등), 동물 바이러스 벡터(레트로바이러스(retrovirus), 아데노바이러스(adenovirus), 백시니아 바이러스(vaccinia virus) 등), 곤충 바이러스 벡터(배큘로바이러스(baculovirus) 등)이 될 수 있다. 상기 발현벡터는 숙주 세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로, 목적에 가장 적합한 숙주세포를 선택하여 사용함이 바람직하다.In the present invention, the type of the expression vector is not particularly limited as long as it can produce the fusion protein of the present invention, but may be preferably plasmid DNA, phage DNA, and more preferably commercially developed Plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc. ), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, vaccinia virus, etc.), insect virus vectors (Baculovirus, etc.). Since the expression vector and the expression level of the protein differ depending on the host cell, it is preferable to select and use the most suitable host cell for the purpose.
본 발명의 일 구체예로, 상기 발현 벡터는 pPT 벡터일 수 있으나, 이에 제한 되는 것은 아니다. In one embodiment of the present invention, the expression vector may be a pPT vector, but is not limited thereto.
본 발명에서 제공하는 형질전환체는 상기 본 발명에서 제공하는 발현벡터를 숙주에 도입하여 형질전환시켜서 제작되고, 상기 발현벡터에 포함된 폴리뉴클레오티드를 발현시켜서, 본 발명의 융합단백질을 생산하는데 사용될 수 있다. The transformant provided by the present invention is produced by introducing the expression vector provided by the present invention into a host and transforming it, and can be used to produce the fusion protein of the present invention by expressing the polynucleotide contained in the expression vector. have.
상기 형질전환은 다양한 방법에 의하여 수행될 수 있는데, 다양한 세포활성을 높은 수준으로 향상시킬 수 있는 효과를 나타내는 본 발명의 융합단백질을 생산할 수 있는 한, 특별히 이에 제한되지 않으나, CaCl2 침전법, CaCl2 침전법에 DMSO(dimethyl sulfoxide)라는 환원물질을 사용함으로써 효율을 높인 Hanahan 방법, 전기천공법(electroporation), 인산칼슘 침전법, 원형질 융합법, 실리콘 카바이드 섬유를 이용한 교반법, 아그로박테리아 매개된 형질전환법, PEG를 이용한 형질전환법, 덱스트란 설페이트, 리포펙타민 및 건조/억제 매개된 형질전환 방법 등이 사용될 수 있다. The transformation can be carried out by various methods, as long as it can produce the fusion protein of the present invention exhibiting the effect of improving various cellular activities to a high level, but is not particularly limited thereto, but the CaCl2 precipitation method, CaCl2 precipitation Hanahan method, which improves efficiency by using a reducing material called DMSO (dimethyl sulfoxide) in the method, electroporation method, calcium phosphate precipitation method, protoplast fusion method, stirring method using silicon carbide fiber, agrobacteria mediated transformation method , Transformation method using PEG, dextran sulfate, lipofectamine, and drying/inhibition mediated transformation method may be used.
또한, 상기 형질전환제의 제작에 사용되는 숙주 역시 본 발명의 융합단백질을 생산할 수 있는 한, 특별히 이에 제한되지 않으나, 대장균(E. coli), 스트렙토마이세스, 살모넬라 티피뮤리움 등의 박테리아 세포; 사카로마이세스 세레비지애, 스키조사카로마이세스 폼베 등의 효모 세포; 피치아 파스토리스 등의 균류 세포; 드로조필라, 스포도프테라 Sf9 세포 등의 곤충 세포; CHO, COS, NSO, 293, 보우 멜라노마 세포 등의 동물 세포; 또는 식물 세포가 될 수 있으나, 이에 제한 되는 것은 아니다. 본 발명의 바람직한 일 구체예로, 상기 형질전환체는 대장균일 수 있으며, 보다 바람직하게는 E. coli JM109일 수 있으나, 이에 제한되는 것은 아니다. 상기 E. coli을 형질전환체로 사용함으로 인하여 치료제로 사용이 가능할 수 있도록 안전하면서도 보다 경제적으로 테리파라타이드를 대량생산 할 수 있는 효과가 있다. In addition, the host used for the preparation of the transformant is also not particularly limited as long as it can produce the fusion protein of the present invention, but is not particularly limited thereto, but bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium; Yeast cells such as Saccharomyces cerevisiae and Schizocaromyces pombe; Fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; Or it may be a plant cell, but is not limited thereto. In a preferred embodiment of the present invention, the transformant may be E. coli, more preferably E. coli JM109, but is not limited thereto. By using the E. coli as a transformant, it is safe and economically effective to mass-produce teriparatide so that it can be used as a therapeutic agent.
본 발명의 일 실시예에서는 상기 테리파라타이드 융합단백질 발현벡터가 도입된 형질전환체로 대장균을 이용하여 본 발명의 목적 단백질인 테리파라타이드의 대량 생산이 가능함을 확인 할 수 있었다. In one embodiment of the present invention, it was confirmed that mass production of teriparatide, the target protein of the present invention, was possible using E. coli as a transformant into which the teriparatide fusion protein expression vector was introduced.
본 발명의 생산방법을 이용할 경우, 기존의 테리파라타이드의 생산방법보다 경제적이면서도 동등 또는 그보다 뛰어난 정제효과를 보이므로, 순도 높은 테리파라타이드를 수득할 수 있어, 대량 생산 방법으로 산업적으로 유용하게 활용될 수 있을 것이다. In the case of using the production method of the present invention, since it is more economical than the conventional production method of teriparatide and exhibits the same or superior purification effect, it is possible to obtain high-purity teriparatide, which is industrially useful as a mass production method. Can be.
도 1은 최종 클로닝한 테리파라타이드 발현 벡터를 모식화한 것이다.
도 2는 시간에 따른 테리파라타이드의 발현 양상을 나타낸 것으로, Lane 1-4 : IPTG 발현 유도 전; Lane 5-9 : IPTG 발현 유도 후 1, 2, 4, 6, 8시간 에 따른 양상을 나타낸 것이며, 화살표는 발현된 teriparatide 융합단백질을 나타낸 것이다.
도 3은 테리파라타이드의 발현 후 세포 파쇄와 워싱에 따른 양상을 나타낸 것으로, Lane 1:발현 후; Lane 2:세포 파쇄 후 펠렛; Lane 3:세포 파쇄 후 상층액; Lane 4:IB 1차 washing후 펠렛; Lane 5:IB 1차 washing후 상층액; Lane 6:IB 2차 washing후 펠렛; Lane 7:IB 2차 washing후 상층액; Lane 8:D.W 1차 washing 후 펠렛; Lane 9:D.W 1차 washing 후 상층액; Lane 10:D.W 2차 washing 후 펠렛; Lane 11:D.W 2차 washing 후 상층액을 나타낸 것이며, 화살표는 테리파라타이드 융합단백질을 나타낸 것이다.
도 4는 친화크로마토그래피를 이용한 1차 정제 크로마토그램 및 SDS-PAGE 분석결과를 나타낸 것으로, 도4A의 경우에는 His-tag 컬럼(친화크로마토그래피)을 이용한 1차 정제 크로마토그램을 나타낸 것이며, 빨간색으로 표시된 상자는 Elution : teriparatide 융합단백질의 피크를 나타낸 것이다.
도 4B의 경우에는 친화크로마토그래피를 이용한 1차 정제 과정의 SDS-PAGE 분석결과를 나타낸 것이다. Lane 1:샘플 로딩시 용해물; Lane 2:샘플 로딩 후 평형화 반응 중 용해물 1; Lane 3:샘플 로딩 후 평형화 반응 중 용해물2; Lane 4:평형화 반응 후 워싱 용해물1; Lane 5:평형화 반응 후 워싱 용해물2; Lane 6-13: 용출된 용해물을 나타내며, 화살표는 테리파라타이드 융합단백질을 나타내는 것이다.
도 5는 HP20 흡착크로마토그래피를 이용한 1차 정제한 결과로, Acetone 농도별 용리액의 SDS-PAGE 분석한 결과이다. Lane 1-3: 10% acetone 버퍼에 의해 용출된 용리액; Lane 4-6: 30% acetone 버퍼에 의해 용출된 용리액; Lane 7-9: 50% acetone 버퍼에 의해 용출된 용리액; Lane 10: 샘플 로딩시 용리액; Lane 11: 샘플 로딩 후 워싱시 용리액; Lane 12-14 : 70% acetone 버퍼에 의해 용출된 용리액; Lane 15-17: 70% acetone 버퍼에 의해 용출된 용리액을 나타낸 것이다.
도 6은 GS20 흡착 크로마토그래피를 이용한 1차 정제한 결과로, Acetone 농도별 용리액의 SDS-PAGE 분석결과를 나타낸 것이다. Lane 1: 샘플 로딩 시 용리액; Lane 2: 샘플 로딩 후 워싱 시 용리액; Lane 3-5: 10% acetone 버퍼에 의해 용출된 용리액; Lane 6-8: 30% acetone 버퍼에 의해 용출된 용리액; Lane 9-11: 50% acetone 버퍼에 의해 용출된 용리액; Lane 12-14: 70% acetone 버퍼에 의해 용출된 용리액; Lane 15-17: 90% acetone 버퍼에 의해 용출된 용리액을 나타낸 것이다.
도 7은 시간별 효소 처리(TEV protease)에 따른 SDS-PAGE 분석 결과를 나타낸 것으로, 효소 처리 0시간부터 4시간까지의 SDS-PAGE 결과이다. Lane 1: 균질화 된 테리파라타이드(histidine이 제거되기 전 상태); Lane 2: 1차 정제된 테리파라타이드; Lane 3: TEV; Lane 4: 효소 처리 0시간; Lane 5: 효소 처리 1시간; Lane 6: 효소 처리 2시간; Lane 7: 효소 처리 3시간; 및 Lane 8: 효소 처리 4시간일 때의 결과를 나타낸 것이다.
도 8은 시간별 효소(TEV protease) 처리에 따른 SDS-PAGE 분석 결과를 나타낸 것이다.
도 8A는 효소 처리 시간과 비율에 따른 SDS-PAGE 결과로, Lane 1: 1차 정제된 테리파라타이드; Lane 2: 효소 처리 1시간 (TEV : TPT = 1:1); Lane 3: 효소 처리 1시간 (TEV : TPT =1:2); Lane 4: 효소 처리 1시간 (TEV : TPT =1:4); Lane 5: 효소 처리 3시간 (TEV : TPT =1:1); Lane 6: 효소 처리 3시간 (TEV : TPT =1:2); Lane 7 : 효소 처리 3시간 (TEV : TPT =1:4); Lane 8: 효소 처리 5시간 (TEV : TPT =1:1); Lane 9 : 효소 처리 5시간 (TEV : TPT =1:2); Lane 10: 효소 처리 5시간 (TEV : TPT =1:4); Lane 11 : 효소 처리 7시간 (TEV : TPT =1:1); Lane 12 : 효소 처리 7시간 (TEV : TPT =1:2); Lane 13 : 효소 처리 7시간 (TEV : TPT =1:4)일 때의 결과를 나타낸 것이다.
도 8B는 효소 처리 시간과 비율에 따른 SDS-PAGE 결과를 나타낸 것이다. Lane 1 : 1차 정제된 테리파라타이드; Lane 2 : 효소 처리 3시간 (TEV : TPT =1:4); Lane 3 : 효소 처리 3시간 (TEV : TPT =1:8); Lane 4 : 효소 처리 3시간 (TEV : TPT =1:12); Lane 5 : 효소 처리 3시간 (TEV : TPT =1:16); Lane 6 : 효소 처리 5시간 (TEV : TPT =1:4); Lane 7 : 효소 처리 5시간 (TEV : TPT =1:8); Lane 8 : 효소 처리 5시간 (TEV : TPT =1:12); Lane 9 : 효소 처리 5시간 (TEV : TPT =1:16); Lane 10 : 효소 처리 7시간 (TEV : TPT =1:4); Lane 11 : 효소 처리 7시간 (TEV : TPT =1:8); Lane 12 : 효소 처리 7시간 (TEV : TPT =1:12); Lane 13 : 효소 처리 7시간 (TEV : TPT =1:16)을 나타낸 결과이다.
도 8C는 효소 처리 시간, 온도, 비율에 따른 SDS-PAGE 결과를 나타낸 것으로,
Lane 1: 1차 정제된 테리파라타이드; Lane 2 : 효소 처리 3시간 (TEV : TPT =1:16) 30℃; Lane 3 : 효소 처리 3시간 (TEV : TPT =1:32) 30℃; Lane 4 : 효소 처리 3시간 (TEV : TPT =1:64) 30℃; Lane 5 : 효소 처리 3시간 (TEV : TPT =1:128) 30℃; Lane 6 : 효소 처리 3시간 (TEV : TPT =1:16) 4℃ ; Lane 7 : 효소 처리 3시간 (TEV : TPT =1:32) 4℃; Lane 8 : 효소 처리 5시간 (TEV : TPT =1:16) 30℃; Lane 9 : 효소 처리 5시간 (TEV : TPT =1:32) 30℃; Lane 10 : 효소 처리 5시간 (TEV : TPT =1:64) 30℃; Lane 11 : 효소 처리 5시간 (TEV : TPT =1:128) 30℃; Lane 12 : 효소 처리 5시간 (TEV : TPT =1:16) 4℃; Lane 13 : 효소 처리 5시간 (TEV : TPT =1:32) 4℃ 일 때의 결과를 나타낸 것이다.
도 8D는 효소 처리 시간, 온도, 비율에 따른 SDS-PAGE 결과를 나타낸 것으로,
Lane 1 : 1차 정제된 테리파라타이드; Lane 2 : 효소 처리 2시간 (TEV : TPT =1:16) 30℃; Lane 3 : 효소 처리 2시간 (TEV : TPT =1:32) 30℃; Lane 4 : 효소 처리 2시간 (TEV : TPT =1:16) 4℃; Lane 5 : 효소 처리 2시간 (TEV : TPT =1:32) 4℃; Lane 6 : 효소 처리 4시간 (TEV : TPT =1:16) 30℃; Lane 7 : 효소 처리 4시간 (TEV : TPT =1:32) 30℃; Lane 8 : 효소 처리 4시간 (TEV : TPT =1:16) 4℃; Lane 9 : 효소 처리 4시간 (TEV : TPT =1:32) 4℃; Lane 10 : 효소 처리 6시간 (TEV : TPT =1:16) 30℃ ; Lane 11 : 효소 처리 6시간 (TEV : TPT =1:32) 30℃; Lane 12 : 효소 처리 6시간 (TEV : TPT =1:16) 4℃; Lane 13 : 효소 처리 6시간 (TEV : TPT =1:32) 4℃일 때의 결과를 나타낸 것이다.
도 9는 이온교환 크로마토그래피를 이용한 2차 정제 크로마토그램 및 SDS-PAGE 와 HPLC 분석결과를 나타낸 것이다.
도 9A는 SP 컬럼을 이용한 2차 정제 크로마토그램을 나타내며, 상자 안의 표시는 Elution : 테리파라타이드의 피크를 나타낸 것이다.
도 9B는 2차 정제 과정의 SDS-PAGE 분석결과를 나타낸 것으로, Lane 1 ; 1차 정제된 전체 용해물; Lane 2 ; 샘플 로딩 시 용해물 1; Lane 3-14 : 2차 정제 시 용출 된 용해물을 나타낸 것이다.
도 9C는 2차 정제된 용해물과 standard teriparatide의 HPLC 분석결과로, STD : standard 0.25mg/ml E8 : 2차 정제된 용해물을 나타낸다.
도 10은 역상크로마토그래피를 이용한 3차 정제결과인 역상컬럼법 정제 크로마토그램 및 SDS-PAGE 분석결과이다.
도 10A는 Reverse-phase 컬럼을 이용한 3차 정제 크로마토그램을 나타낸 것이며, 상자 안 Elution : teriparatide의 피크를 나타낸 것이고, 도10B는 3차 정제 과정의 SDS-PAGE 분석결과이다. Lane 1-14 : 3차 정제 시 용출 된 용해물을 나타내며, 화살표는 테리파라타이드를 나타내는 것이다. 도10C는 3차 정제된 용해물과 standard teriparatide의 HPLC 분석결과이다. STD : standard 0.125mg/ml E : 3차 정제된 용해물을 나타낸다.
도 11은 정제된 테리파라타이드의 MALDI-TOF 분석결과를 나타낸 것이다. 1 is a schematic diagram of the final cloned teriparatide expression vector.
Figure 2 shows the expression pattern of teriparatide over time, Lane 1-4: before IPTG expression induction; Lane 5-9: It shows the pattern according to 1, 2, 4, 6, 8 hours after induction of IPTG expression, and the arrows indicate the expressed teriparatide fusion protein.
3 is a diagram showing the pattern according to cell disruption and washing after expression of teriparatide, Lane 1: after expression; Lane 2: pellet after cell disruption; Lane 3: supernatant after cell disruption; Lane 4: IB pellet after the first washing; Lane 5: IB supernatant after the first washing; Lane 6: Pellets after the second IB washing; Lane 7: IB supernatant after second washing; Lane 8: pellet after the first washing of DW; Lane 9: Supernatant after first washing of DW; Lane 10: pellets after the second washing of DW; Lane 11: shows the supernatant after the second washing of DW, and the arrow shows the teriparatide fusion protein.
Figure 4 shows the results of the first purification chromatogram and SDS-PAGE analysis using affinity chromatography, and in the case of Figure 4A, the first purification chromatogram using a His-tag column (affinity chromatography) is shown, in red. The box indicated is the peak of the Elution: teriparatide fusion protein.
4B shows the SDS-PAGE analysis result of the first purification process using affinity chromatography. Lane 1: Lysate upon sample loading; Lane 2:
5 is a result of primary purification using HP20 adsorption chromatography, and is a result of SDS-PAGE analysis of the eluent for each concentration of acetone. Lane 1-3: eluent eluted with 10% acetone buffer; Lane 4-6: eluent eluted with 30% acetone buffer; Lane 7-9: eluent eluted with 50% acetone buffer; Lane 10: eluent at sample loading; Lane 11: Eluent upon washing after sample loading; Lane 12-14: eluent eluted with 70% acetone buffer; Lane 15-17: Shows the eluent eluted with 70% acetone buffer.
6 is a result of primary purification using GS20 adsorption chromatography, and shows the results of SDS-PAGE analysis of the eluent for each concentration of acetone. Lane 1: eluent upon sample loading; Lane 2: eluent for washing after sample loading; Lane 3-5: eluent eluted with 10% acetone buffer; Lane 6-8: eluent eluted with 30% acetone buffer; Lane 9-11: eluent eluted with 50% acetone buffer; Lane 12-14: eluent eluted with 70% acetone buffer; Lane 15-17: shows the eluent eluted with 90% acetone buffer.
7 shows the results of SDS-PAGE analysis according to the time-dependent enzyme treatment (TEV protease), and shows the SDS-PAGE results from 0 hours to 4 hours of enzyme treatment. Lane 1: Homogenized teriparatide (before histidine was removed); Lane 2: primary purified teriparatide; Lane 3: TEV; Lane 4: enzyme treatment 0 hours; Lane 5:
8 shows the results of SDS-PAGE analysis according to the time-dependent enzyme (TEV protease) treatment.
Figure 8A is a result of SDS-PAGE according to the enzyme treatment time and ratio, Lane 1: primary purified teriparatide; Lane 2:
Figure 8B shows the SDS-PAGE result according to the enzyme treatment time and ratio. Lane 1: primary purified teriparatide; Lane 2:
Figure 8C shows the SDS-PAGE results according to the enzyme treatment time, temperature, and ratio,
Lane 1: primary purified teriparatide; Lane 2: enzyme treatment 3 hours (TEV: TPT = 1:16) 30°C; Lane 3: Enzyme treatment 3 hours (TEV: TPT = 1:32) 30°C; Lane 4: Enzyme treatment 3 hours (TEV: TPT = 1:64) 30°C; Lane 5: Enzyme treatment 3 hours (TEV: TPT = 1:128) 30°C; Lane 6: Enzyme treatment 3 hours (TEV: TPT = 1:16) 4°C; Lane 7: Enzyme treatment 3 hours (TEV: TPT = 1:32) 4°C; Lane 8: enzyme treatment 5 hours (TEV: TPT = 1:16) 30°C; Lane 9: enzyme treatment 5 hours (TEV: TPT = 1:32) 30°C; Lane 10: enzyme treatment for 5 hours (TEV: TPT = 1:64) 30°C; Lane 11: enzyme treatment 5 hours (TEV: TPT = 1:128) 30°C; Lane 12: 5 hours of enzyme treatment (TEV: TPT = 1:16) 4°C; Lane 13: Enzyme treatment 5 hours (TEV: TPT = 1:32) shows the results at 4 ℃.
Figure 8D shows the SDS-PAGE results according to the enzyme treatment time, temperature, and ratio,
Lane 1: primary purified teriparatide; Lane 2: enzyme treatment 2 hours (TEV: TPT = 1:16) 30°C; Lane 3: Enzyme treatment 2 hours (TEV: TPT = 1:32) 30°C; Lane 4: enzyme treatment 2 hours (TEV: TPT = 1:16) 4°C; Lane 5: Enzyme treatment 2 hours (TEV: TPT = 1:32) 4°C; Lane 6: Enzyme treatment 4 hours (TEV: TPT = 1:16) 30°C; Lane 7: Enzyme treatment 4 hours (TEV: TPT = 1:32) 30°C; Lane 8: 4 hours of enzyme treatment (TEV: TPT = 1:16) 4°C; Lane 9: Enzyme treatment 4 hours (TEV: TPT = 1:32) 4°C; Lane 10: 6 hours of enzyme treatment (TEV: TPT = 1:16) 30°C; Lane 11: Enzyme treatment 6 hours (TEV: TPT = 1:32) 30°C; Lane 12: 6 hours of enzyme treatment (TEV: TPT = 1:16) 4°C; Lane 13: Enzyme treatment 6 hours (TEV: TPT = 1:32) shows the results at 4 ℃.
9 shows the results of secondary purification chromatogram and SDS-PAGE and HPLC analysis using ion exchange chromatography.
9A shows a second purification chromatogram using an SP column, and the mark in the box shows the peak of Elution: teriparatide.
9B shows the results of SDS-PAGE analysis of the second purification process,
9C shows the result of HPLC analysis of the second purified lysate and standard teriparatide, STD: standard 0.25mg/ml E8: secondly purified lysate.
10 is a reverse-phase column method purification chromatogram and SDS-PAGE analysis results, which are the third purification results using reverse phase chromatography.
FIG. 10A shows a third purification chromatogram using a reverse-phase column, and shows the peak of Elution: teriparatide in the box, and FIG. 10B is a SDS-PAGE analysis result of the third purification process. Lane 1-14: indicates the eluted lysate during the third purification, and the arrow indicates teriparatide. Figure 10C shows the results of HPLC analysis of the third purified lysate and standard teriparatide. STD: standard 0.125mg/ml E: represents the third purified lysate.
11 shows the results of MALDI-TOF analysis of purified teriparatide.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실시예 1: 생산균주 및 플라스미드 제작Example 1: Production strain and plasmid production
대장균 JM109를 사용하여, pPT 벡터에 테리파라타이드 융합단백질의(서열번호 1) 서열 및 암피실린 저항 유전자와 histidine tagging site를 첨가시켜 클로닝하였다. pPT 벡터에 삽입된 융합단백질의 유전자서열은 서열번호 1와 같으며, 그 결과 도 1과 같은 벡터를 제작하였다. 건설된 벡터의 이름은 pPT-TPT라고 명명하였다. Using E. coli JM109, the pPT vector was cloned by adding the sequence of the teriparatide fusion protein (SEQ ID NO: 1), the ampicillin resistance gene and the histidine tagging site. The gene sequence of the fusion protein inserted into the pPT vector was the same as SEQ ID NO: 1, and as a result, a vector as shown in FIG. 1 was produced. The constructed vector was named pPT-TPT.
서열번호1: ATGACCATGATTACGAATTCCCCGGAGATCTCCCACCACCACCACCACCACCACCACCACCACCAGCTGATCTCAGAAGCTCGTGAAAACCTGTACTTCCAGAGCGTGAGCGAAATCCAGCTGATGCATAACCTGGGCAAACACCTGAACAGCATGGAACGTGTGGAATGGCTGCGTAAAAAACTGCAGGATGTGCACAACTTCSEQ ID NO: 1: ATGACCATGATTACGAATTCCCCGGAGATCTCCCACCACCACCACCACCACCACCACCACCACCAGCTGATCTCAGAAGCTCGTGAAAACCTGTACTTCCAGAGCGTGAGCGAAATCCAGCTGATGCATAACCTGGTCGCAAACACCTGAACAGCATGGAACGTGTGGAATGCGATGGCGTAACT
실시예 2: 테리파라타이드(teriparatide)의 발현을 위한 발효공정Example 2: Fermentation process for expression of teriparatide
테리파라타이드 발현을 위한 pPT-TPT 벡터가 삽입된 E. coli JM109/pPT-TPT 발현균주를 암피실린(50ug/ml)이 포함된 LB 배지에서 37℃ 온도로 16시간 동안 전 배양 한 후, 고밀도배양을 위하여 1L modified LB 배지에 접종하였다. 배양의 세부적인 조건은 pH 6.8, pO2 30%로 유지시켰다. pH 변화에 따라 50% 포도당을 탄소원으로 점진적으로 첨가하였으며, 질소원으로써 동일한 양의 35% yeast extract도 함께 공급하였다. 최종적으로 테리파라타이드를 발현시키기 위하여 1 mM의 IPTG를 접종하였고 그 후 8시간 배양하여 발현을 유도하였다.The E. coli JM109/pPT-TPT expressing strain inserted with the pPT-TPT vector for teriparatide expression was pre-incubated in LB medium containing ampicillin (50 ug/ml) for 16 hours at 37°C for 16 hours, followed by high-density culture. For this, it was inoculated in 1L modified LB medium. The detailed conditions of the culture were maintained at pH 6.8 and pO 2 30%. According to the pH change, 50% glucose was gradually added as a carbon source, and the same amount of 35% yeast extract was also supplied as a nitrogen source. Finally, in order to express teriparatide, 1 mM of IPTG was inoculated and then cultured for 8 hours to induce expression.
시간에 따른 발현 수준을 확인하기 위해 일정 시간 간격으로 샘플을 채취하여 흡광도를 측정하였다. 배양 22시간 후 흡광도가 28.7에 도달 하였을 때 IPTG의 최종 농도가 1mM이 되도록 처리하여 발현을 유도하였다. 이후 8시간 동안 더 배양한 후 최종 46.8 흡광도 값에 도달하여 종료하였으며 1L 배양 배지에서 54.18g의 세포를 회수하였다. 그 결과 도 2와 같이 SDS-PAGE 결과로 시간에 따른 테리파라타이드의 발현양상을 확인할 수 있었다. In order to check the expression level over time, samples were taken at regular time intervals and absorbance was measured. When the absorbance reached 28.7 after 22 hours of culture, the final concentration of IPTG was 1 mM to induce expression. After further culturing for 8 hours, the final absorbance value of 46.8 was reached and terminated, and 54.18 g of cells were recovered in 1L culture medium. As a result, it was possible to confirm the expression pattern of teriparatide over time as a result of SDS-PAGE as shown in FIG. 2.
도 2에서 볼 수 있듯이, lane4번에서 IPTG를 첨가 한 후 발현이 잘 된 것을 확인하였다. 발현된 teriparatide 융합단백질의 크기는 약 8 kDa으로 PAGE 결과상 근접한 사이즈 근처에서 밴드가 확인이 되었고 원하는 단백질이 약 14% 정도로 발현이 되었음을 확인할 수 있었다. As can be seen in Figure 2, after adding IPTG in
실시예 3: 세포 파쇄와 Inclusion body의 분리 정제Example 3: Cell disruption and separation and purification of inclusion body
배양된 세포 용액을 원심분리(12,000 rpm, 20 min, 4℃)한 후 침전물을 lysis 용액(10% Sucrose, 10 mM Tris, 50 mM EDTA, 0.2M NaCl, pH7.9)에 녹였다. 이 용액을 Homogenizer(800bar)를 이용하여 세포를 파쇄한 후 원심분리(12000 rpm, 20 min, 4℃)를 수행하였다. 이렇게 하여 얻어진 Inclusion body를 washing buffer(20 mM Tris, 1 mM EDTA, 0.02% Lysozyme, 1% TritonX-100, 0.5 M Urea, pH7.0)와 증류수로 세척하였다.After centrifuging the cultured cell solution (12,000 rpm, 20 min, 4°C), the precipitate was dissolved in a lysis solution (10% Sucrose, 10 mM Tris, 50 mM EDTA, 0.2M NaCl, pH7.9). This solution was subjected to centrifugation (12000 rpm, 20 min, 4° C.) after crushing the cells using a homogenizer (800 bar). The inclusion body thus obtained was washed with a washing buffer (20 mM Tris, 1 mM EDTA, 0.02% Lysozyme, 1% TritonX-100, 0.5 M Urea, pH 7.0) and distilled water.
세척 후 회수된 Inclusion body를 용액(8 M Urea, 20 mM Tris, pH8.0)으로 녹였다. 이 용액을 상온에서 교반하면서 16시간 이상 반응시켜 세포의 균질화 과정을 거쳤다.After washing, the recovered inclusion body was dissolved in a solution (8 M Urea, 20 mM Tris, pH 8.0). The solution was stirred at room temperature and reacted for 16 hours or longer to homogenize the cells.
그 결과로, 세포 파쇄 후 Inclusion body형태로 최종 무게 18.81 g이 회수되었다. 파쇄 후 pellet과 상층액을 수거하여 SDS-PAGE을 통해 확인하였고, 도 3과 같이 발현 후의 세포파쇄와 워싱에 따른 양상을 나타냄을 확인할 수 있었다. 도 3에서 볼 수 있듯이, pellet에서 teriparatide의 band가 나타난 것으로 보아 Inclusion body form인 것을 확인 할 수 있었다. 그 후 Inclusion body washing buffer과 증류수를 통해 각각 2차례 정제를 진행하였고 최종 무게 7 g를 회수하였다. As a result, the final weight of 18.81 g was recovered in the form of an inclusion body after cell disruption. After crushing, the pellet and the supernatant were collected and confirmed through SDS-PAGE, and as shown in FIG. 3, it was confirmed that the cell lysis and washing after expression were shown. As can be seen in FIG. 3, it was confirmed that the band of teriparatide appeared in the pellet, indicating that it was in inclusion body form. After that, purification was performed twice each through the inclusion body washing buffer and distilled water, and a final weight of 7 g was recovered.
실시예 4. 흡착크로마토그래피를 이용한 1차 정제 Example 4. Primary purification using adsorption chromatography
4-1. 1차 정제 (His-tag affinity chromatography)와의 비교4-1. Comparison with primary purification (His-tag affinity chromatography)
기존의 단백질 정제에서 사용하였던 His-tag 이용한 친화크로마토그래피와의 결과를 비교하기 위하여 균질화 시킨 용액을 his-tag column을 이용하여 1차 정제를 실시하였다. Stripping buffer(20 mM Sodium phosphate, 0.5 M NaCl, 50 mM EDTA, pH7.4)로 컬럼의 불순물을 제거 하였고, Binding buffer(20 mM Tris, 0.5 M NaCl, 5% Glycerol, pH8.0)으로 column을 안정화시켰다. 그 후 샘플을 flow rate 2 ml/min을 이용하여 흘려주었고 Elution buffer(20 mM Tris, 0.5 M NaCl, 5% Glycerol, 1 M Imidazole, pH8.0)을 이용하여 용출 하였다. SDS-PAGE를 이용하여 발현을 확인하였으며 그 결과는 도 4에 도시하였다. In order to compare the results with the affinity chromatography using His-tag used in the conventional protein purification, the homogenized solution was subjected to primary purification using a his-tag column. Impurities from the column were removed with a stripping buffer (20 mM Sodium phosphate, 0.5 M NaCl, 50 mM EDTA, pH 7.4), and the column was opened with a binding buffer (20 mM Tris, 0.5 M NaCl, 5% Glycerol, pH 8.0). Stabilized. Thereafter, the sample was flowed at a flow rate of 2 ml/min, and elution buffer (20 mM Tris, 0.5 M NaCl, 5% Glycerol, 1 M Imidazole, pH 8.0) was used to elution. Expression was confirmed using SDS-PAGE, and the results are shown in FIG. 4.
도 4에서 볼 수 있듯이, 균질화 시킨 샘플을 컬럼 내에 로딩하고 1 M Imidazole이 포함된 용리버퍼로 용리시킨 결과 피크 형성을 확인함으로서, 테리파라타이드(teriparatide) 융합단백질을 확인할 수 있었으며 (도 4A), 수거된 용리액을 SDS-PAGE로 분석한 결과, 대부분의 불순물이 제거되고 1차 정제된 테리파라타이드 융합단백질을 얻을 수 있었다(도 4B). As can be seen in Figure 4, by loading the homogenized sample into the column and eluting with an elution buffer containing 1 M Imidazole, the peak formation was confirmed, thereby confirming the teriparatide fusion protein (Figure 4A), As a result of analyzing the collected eluent by SDS-PAGE, most of the impurities were removed and a primary purified teriparatide fusion protein was obtained (FIG. 4B).
4-2. 1차 정제 (HP 흡착 chromatography)4-2. 1st purification (HP adsorption chromatography)
상기 실시예 3에서 균질화 시킨 용액을 HP20이 충전된 column을 이용하여 정제를 실시하였다. Equilibrium buffer(0.1 M Ethylenediamine, pH 10)을 사용하여 flow rate 5 ml/min의 속도로 컬럼을 안정화시켰다. 그 후 샘플을 flow rate 1ml/min을 이용하여 흘려주었고, Washing buffer(20 mM Ethylenediamine, pH 10)을 불순물들을 제거하였다. Elution buffer(20 mM Ethylenediamine, 10%,30%,50%,90% Acetone, pH10)을 사용하여 용출하였다. 각각의 용출액을 수거하여 SDS-PAGE를 이용하여 발현을 확인하였고 그 결과는 도 5와 같다. The solution homogenized in Example 3 was purified using a column filled with HP20. Equilibrium buffer (0.1 M Ethylenediamine, pH 10) was used to stabilize the column at a flow rate of 5 ml/min. Thereafter, the sample was flowed at a flow rate of 1 ml/min, and impurities were removed in a washing buffer (20 mM Ethylenediamine, pH 10). Elution buffer (20 mM Ethylenediamine, 10%, 30%, 50%, 90% Acetone, pH 10) was used to elution. Each eluate was collected, and expression was confirmed using SDS-PAGE, and the results are shown in FIG. 5.
도 5에서 볼 수 있듯이, Acetone의 농도별 퍼센트로 포함된 버퍼로 용리시킨 결과 대부분의 불순물이 제거 되고 teriparatide가 정제되었음을 SDS-PAGE를 통해 확인 할 수 있었다. 또한, 아세톤의 농도가 50%에서 원하는 단백질의 밴드가 진하게 나타났고, 70%에서도 밴드가 흐리게 나타나는 것을 확인 할 수 있었다. As can be seen in FIG. 5, it was confirmed through SDS-PAGE that most of the impurities were removed and teriparatide was purified as a result of elution with a buffer containing a percentage of acetone concentration. In addition, when the concentration of acetone was 50%, the band of the desired protein appeared dark, and it was confirmed that the band was blurred even at 70%.
4-3. 1차 정제 (GS20 흡착 chromatography)4-3. First purification (GS20 adsorption chromatography)
상시 실시예 3에서 균질화 시킨 용액을 GS20이 충전된 column을 이용하여 정제를 실시하였다. Equilibrium buffer(0.1 M Ethylenediamine, pH 8)을 사용하여 flow rate 5 ml/min의 속도로 컬럼을 안정화시켰다. 그 후 샘플을 flow rate 1ml/min을 이용하여 흘려주었고, Washing buffer(20 mM Ethylenediamine, pH 10)을 불순물들을 제거하였다. Elution buffer(20 mM Ethylenediamine, 10%,30%,50%,90% Acetone, pH10)을 사용하여 용출하였다. 각각의 용출액을 수거하여 SDS-PAGE를 이용하여 발현을 확인하였다. 그 결과는 도 6에 도시하였다. The solution homogenized in Example 3 at all times was purified using a column filled with GS20. Equilibrium buffer (0.1 M Ethylenediamine, pH 8) was used to stabilize the column at a flow rate of 5 ml/min. Thereafter, the sample was flowed at a flow rate of 1 ml/min, and impurities were removed in a washing buffer (20 mM Ethylenediamine, pH 10). Elution buffer (20 mM Ethylenediamine, 10%, 30%, 50%, 90% Acetone, pH 10) was used to elution. Each eluate was collected and expression was confirmed using SDS-PAGE. The results are shown in FIG. 6.
도 6에서 볼 수 있듯이, Acetone의 농도별 퍼센트로 포함된 버퍼로 용리 시킨 결과 대부분의 불순물이 제거 되고 teriparatide가 정제되었음을 SDS-PAGE를 통해 확인 할 수 있었다. 아세톤의 농도가 50%에서 원하는 단백질의 밴드가 진하게 나타났고, 70%에서도 밴드가 흐리게 나타나는 것을 또한 확인 하였다. As can be seen in FIG. 6, as a result of eluting with a buffer containing a percentage of acetone concentration, it was confirmed through SDS-PAGE that most of the impurities were removed and teriparatide was purified. When the concentration of acetone was 50%, the band of the desired protein appeared dark, and it was also confirmed that the band was blurred even at 70%.
실시예 5. 효소 반응을 통한 융합단백질의 절단Example 5. Cleavage of fusion protein through enzymatic reaction
상기 실시예 4에서 1차 정제된 융합단백질에서 테리파라타이드를 절단 후 정제하기 위하여 TEV(Tobacco etch virus) protease를 사용하였다. TEV의 양과 균질화 된 teriparatide의 단백질양을 1:1의 비율로 섞어 상온에서 진행하였다. 0시간, 6시간, 8시간 진행하였으며 시간별로 HPLC 분석을 확인하였다. 효소반응을 총 8시간을 수행하여 시간대별로 시료를 채취하여 SDS-PAGE 분석을 수행하였으며 결과는 도 7에 도시하였다. 상기 도 7의 SDS-PAGE 분석 결과에 의하면 상온에서 4시간을 최적의 반응조건으로 확인할 수 있었다.TEV (Tobacco etch virus) protease was used to purify teriparatide after cleavage from the first purified fusion protein in Example 4. The amount of TEV and the amount of homogenized teriparatide protein were mixed in a ratio of 1:1 and proceeded at room temperature. It proceeded for 0 hours, 6 hours, and 8 hours, and HPLC analysis was confirmed by time. The enzymatic reaction was performed for a total of 8 hours, samples were collected for each time period, and SDS-PAGE analysis was performed, and the results are shown in FIG. 7. According to the SDS-PAGE analysis result of FIG. 7, 4 hours at room temperature could be confirmed as the optimal reaction condition.
또한, 1차 정제된 테리파라타이드 융합단백질의 최적의 효소 처리 방법을 알아보기 위해 온도, 시간, 비율에 대한 조건 실험을 진행하였다. In addition, in order to find out the optimal enzyme treatment method of the first purified teriparatide fusion protein, conditions for temperature, time, and ratio were tested.
(A) TEV : TPT의 단백질양 비율을 1:1, 1:2, 1:4로 30℃에서 진행하였으며, 7시간 효소 반응을 실시하였다.(A) TEV: The protein amount ratio of TPT was 1:1, 1:2, 1:4, and proceeded at 30°C, and the enzyme reaction was carried out for 7 hours.
(B) TEV : TPT의 단백질양 비율을 1:4, 1:8, 1:12, 1:16로 30℃에서 진행하였으며, 7시간 효소 반응을 실시하였다.(B) TEV: The protein amount ratio of TPT was 1:4, 1:8, 1:12, 1:16, and proceeded at 30°C, and the enzyme reaction was carried out for 7 hours.
(C) TEV : TPT의 단백질양 비율을 1:1, 1:2, 1:4, 1:8, 1:12, 1:16, 1:32, 1:64, 1:128로 4℃와 30℃에서 진행하였으며, 5시간 효소 반응을 실시하였다.(C) TEV: TPT protein amount ratio of 1:1, 1:2, 1:4, 1:8, 1:12, 1:16, 1:32, 1:64, 1:128 at 4℃ and It proceeded at 30° C., and enzymatic reaction was performed for 5 hours.
(D) TEV : TPT의 단백질양 비율을 1:16, 1:32로 4℃와 30℃로 진행하였으며, 계속 효소를 주입하여 6시간 효소 반응을 실시하였다.(D) TEV: The protein amount ratio of TPT was 1:16, 1:32, and proceeded to 4℃ and 30℃, and the enzyme was continuously injected to carry out the enzyme reaction for 6 hours.
시간 마다 샘플링을 하였으며, pH를 3.0으로 맞춰서 효소 처리 반응 종료하였다. 그 후 SDS-PAGE를 통해서 확인하였으며 그 결과는 도 8에 도시하였다. Sampling was performed every time, and the enzyme treatment reaction was terminated by adjusting the pH to 3.0. Then, it was confirmed through SDS-PAGE, and the results are shown in FIG. 8.
도 8에서 볼 수 있듯이, 도 8A는 TEV : TPT의 단백질양 비율을 1:1, 1:2, 1:4로 30℃에서 진행한 결과이다. SDS-PAGE 결과 상 1:4의 비율 5시간에서 진하게 나타난 것으로 보아 최적의 비율은 1:4이고, 최적의 반응 시간은 5시간 반응이 가장 효소 처리가 잘 되었음을 확인할 수 있었다. As can be seen in Figure 8, Figure 8A is a result of proceeding at 30 ℃ TEV: TPT protein amount ratio of 1:1, 1:2, 1:4. As a result of SDS-PAGE, the ratio of 1:4 was found to be dark at 5 hours, indicating that the optimum ratio was 1:4, and the reaction time of 5 hours was the best enzyme treatment.
도 8B는 TEV : TPT의 단백질양 비율을 1:4, 1:8, 1:12, 1:16로 30℃에서 진행한 결과로 SDS-PAGE 결과 상 단백질 1:16의 비율에서 잘려진 teriparatide를 확인할 수 있었다. FIG. 8B shows teriparatide cut at the ratio of protein 1:16 on the SDS-PAGE result as a result of proceeding at 30° C. with the protein amount ratio of TEV: TPT 1:4, 1:8, 1:12, 1:16 Could
도 8C는 TEV : TPT의 단백질양 비율을 1:1, 1:2, 1:4, 1:8, 1:12, 1:16, 1:32, 1:64, 1:128로 진행한 결과로, SDS-PAGE 결과 상 단백질 1:128의 비율에서 잘려진 teriparatide가 확인하였으며, 4℃에서도 효소가 활성을 보여 teriparatide가 잘려진 것을 확인할 수 있었다. Figure 8C shows the results of progressing the protein amount ratio of TEV: TPT to 1:1, 1:2, 1:4, 1:8, 1:12, 1:16, 1:32, 1:64, 1:128 As a result of SDS-PAGE, teriparatide, which was cut at the ratio of protein 1:128, was confirmed, and the enzyme was active even at 4°C, indicating that teriparatide was cut.
도 8D는 TEV : TPT의 단백질양 비율을 1:16, 1:32로 4℃와 30℃로 진행한 결과로, 효소를 계속 주입하였을 때 1:16의 비율로 30℃에서 4~5시간 사이에서 최적의 효소 처리를 보임을 확인할 수 있었다. Figure 8D is a result of proceeding to 4 ℃ and 30 ℃ TEV: TPT protein amount ratio of 1:16, 1:32, when the enzyme was continuously injected at 30 ℃ between 4 to 5 hours at a ratio of 1:16 It was confirmed that the optimal enzyme treatment was shown.
실시예 6. 2차 정제 (이온교환수지 컬럼법) Example 6. Secondary purification (ion exchange resin column method)
1차 정제 시킨 용액을 SDS-PAGE로 확인한 결과 원하는 단백질의 밴드가 보이는 용출액을 수거하여 SP cation exchange column을 이용하여 2차 정제를 실시하였다. B buffer(7 M Urea, 0.25 M Acetic acid, 1 M NaCl, pH2.5)를 이용하여 column의 불순물을 제거 하였고, A buffer(7 M Urea, 0.25 M Acetic acid, pH2.5)를 이용하여 컬럼을 안정화시켰다. 샘플을 flow rate 1ml/min으로 이용하여 로딩하였고, Elution은 B buffer의 퍼센트를 0~100%까지 변화를 주어 100분 동안 분리 정제하였다. 그 결과는 도 9에 도시하였다. As a result of confirming the first purified solution by SDS-PAGE, the eluate showing the desired protein band was collected, and the second purification was performed using an SP cation exchange column. B buffer (7 M Urea, 0.25 M Acetic acid, 1 M NaCl, pH 2.5) was used to remove impurities from the column, and A buffer (7 M Urea, 0.25 M Acetic acid, pH 2.5) was used to remove the column. Stabilized. Samples were loaded using a flow rate of 1 ml/min, and Elution was separated and purified for 100 minutes by changing the percentage of B buffer from 0 to 100%. The results are shown in FIG. 9.
샘픔을 컬럼 내 로딩하고 1M NaCl이 포함된 용리 버퍼로 용리 시킨 결과 세 가지의 피크를 형성 하였으며(도 9A), 각각의 용리액을 SDS-PAGE로 분석한 결과를 도 9B에 도시하였다. 정제된 teriparatide의 순도를 확인하기 위하여 HPLC 분석을 시행하였다. 분석 결과 18.83분에 용리 되었으며, standard teriparatide의 분석 결과 머무름 시간이 일치하는 것을 확인할 수 있었다. As a result of loading the sample into the column and eluting with an elution buffer containing 1M NaCl, three peaks were formed (FIG. 9A), and the results of analyzing each eluent by SDS-PAGE are shown in FIG. 9B. HPLC analysis was performed to confirm the purity of the purified teriparatide. As a result of the analysis, it was eluted at 18.83 minutes, and as a result of the analysis of standard teriparatide, it was confirmed that the retention time was consistent.
실시예 7. 3차 정제 (역상컬럼법)Example 7. 3rd purification (reverse phase column method)
상기 실시예 6에서 2차 정제 시킨 용액을 Reverse phase column을 이용하여 3차 정제를 실시하였다. 상기 정제는 2차 정제 후 SDS-PAGE로 확인한 결과와 HPLC분석결과 teriparatide 밴드가 보이는 용출액을 수거하여 다음 3차 정제로 C-18 컬럼을 사용하여 역상컬럼법으로 진행하였다. 100% ACN으로 column의 불순물을 제거 하였고, A buffer (0.1% TFA, DW)를 사용하여 column을 안정화시켰다. flow rate 1 ml/min으로 샘플을 로딩하였다. 그 후 B buffer(0.1% TFA, 50% ACN, DW)를 사용하여 ACN의 퍼센트를 선형적으로 변화를 주어 60 min동안 분리 정제 하였다. 샘플을 컬럼 내 로딩하고 ACN이 포함된 용리 버퍼로 용리 시킨 결과 도 10과 같은 크로마토그램을 형성함을 확인할 수 있었다. 도 10에서 볼 수 있듯이, 빨간 박스로 표시된 부분이 teriparatide이며(도10A), 각각의 용리액을 SDS-PAGE로 분석한 결과는 도10B에 도시하였다. The solution secondarily purified in Example 6 was subjected to third purification using a Reverse phase column. The purification was performed by a reversed-phase column method using a C-18 column as a result of the second purification and then collecting the eluate showing the teriparatide band as a result of SDS-PAGE and HPLC analysis. Impurities from the column were removed with 100% ACN, and the column was stabilized using A buffer (0.1% TFA, DW). Samples were loaded at a flow rate of 1 ml/min. After that, the percentage of ACN was linearly changed using B buffer (0.1% TFA, 50% ACN, DW) to separate and purify for 60 min. As a result of loading the sample into the column and eluting with an elution buffer containing ACN, it was confirmed that a chromatogram as shown in FIG. 10 was formed. As can be seen in FIG. 10, the area marked with a red box is teriparatide (FIG. 10A), and the results of analyzing each eluent by SDS-PAGE are shown in FIG. 10B.
상기 3차 정제로 인하여 불순물이 제거된 테리파라타이드를 수득할 수 있음을 확인하였다. It was confirmed that teriparatide from which impurities were removed can be obtained by the third purification.
실시예 8. 분자량 분석을 통한 테리파라타이드의 정체 규명Example 8. Identification of the identity of teriparatide through molecular weight analysis
상기 실시예에서 발현 및 정제를 통하여 획득한 테리파라타이드의 정체를 규명하기 위하여 MALDI-TOF 분석법을 이용하여 mass 분석을 실시하였다.In order to identify the identity of teriparatide obtained through expression and purification in the above example, mass analysis was performed using the MALDI-TOF assay.
정제된 teriparatide의 분자량은 MALDI-TOF 분석법으로 확인하였으며 그 결과는 도 11에 도시하였다. 화살표로 표시된 피크가 정제된 teriparatide의 피크로 예측되며, 측정된 분자량은 4.121kDa으로 예측된 분자량인 4.117kDa과 거의 일치 하였다.The molecular weight of the purified teriparatide was confirmed by MALDI-TOF analysis, and the results are shown in FIG. 11. The peak indicated by the arrow was predicted as the peak of purified teriparatide, and the measured molecular weight was almost identical to the predicted molecular weight of 4.117 kDa, 4.121 kDa.
따라서, 상기와 같은 결과로 본 발명의 제조방법에 의하여 불순물이 제거된 테리파라타이드를 수득할 수 있음을 확인할 수 있었다. Accordingly, it was confirmed that teriparatide from which impurities were removed can be obtained by the production method of the present invention as a result of the above.
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 be able to 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 thereof, are included in the scope of the present invention.
<110> Sunchon National University Industry-academic Cooperation Foundation <120> The mass production process of teriparatide using E.coli <130> KPA190438-KR-P1 <150> KR 10-2019-0041758 <151> 2019-04-10 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> fusion protein <400> 1 atgaccatga ttacgaattc cccggagatc tcccaccacc accaccacca ccaccaccac 60 caccagctga tctcagaagc tcgtgaaaac ctgtacttcc agagcgtgag cgaaatccag 120 ctgatgcata acctgggcaa acacctgaac agcatggaac gtgtggaatg gctgcgtaaa 180 aaactgcagg atgtgcacaa cttc 204 <110> Sunchon National University Industry-academic Cooperation Foundation <120> The mass production process of teriparatide using E.coli <130> KPA190438-KR-P1 <150> KR 10-2019-0041758 <151> 2019-04-10 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> fusion protein <400> 1 atgaccatga ttacgaattc cccggagatc tcccaccacc accaccacca ccaccaccac 60 caccagctga tctcagaagc tcgtgaaaac ctgtacttcc agagcgtgag cgaaatccag 120 ctgatgcata acctgggcaa acacctgaac agcatggaac gtgtggaatg gctgcgtaaa 180 aaactgcagg atgtgcacaa cttc 204
Claims (10)
(b) 상기 배양된 형질전환체, 이의 배양물 또는 이의 파쇄물을 흡착 크로마토그래피에 적용하여 테라파라타이드 융합단백질을 분리하는 1차 정제단계;
(c) 상기 제1차 정제단계에서 수득된 융합단백질에 TEV(Tobacco etch virus) 단백질분해효소를 처리하는 단계;
(d) 상기 단백질분해효소 처리 후 절단된 테리파라타이드를 이온교환수지 컬럼 크로마토그래피를 이용하여 분리하는 2차 정제단계; 및
(e) 상기 분리된 테리파라타이드를 역상 컬럼 크로마토그래피로 정제하는 3차 정제단계를 포함하는, 테리파라타이드 대량 생산방법.
(a) culturing a transformant into which an expression vector containing a polynucleotide encoding a teriparatide fusion protein has been introduced;
(b) a first purification step of separating the teraparatide fusion protein by applying the cultured transformant, its culture or its lysate to adsorption chromatography;
(c) treating the fusion protein obtained in the first purification step with a TEV (Tobacco etch virus) protease;
(d) a second purification step of separating teriparatide cut after the protease treatment by using ion exchange resin column chromatography; And
(e) comprising a third purification step of purifying the separated teriparatide by reverse phase column chromatography, teriparatide mass production method.
The method of claim 1, wherein the teriparatide fusion protein is encoded by the nucleotide sequence of SEQ ID NO: 1.
The method of claim 1, wherein the transformant is E. coli.
(b1) 상기 배양된 형질전환체, 이의 배양물 또는 이의 파쇄물에서 봉입체 형태의 융합단백질을 수득하는 단계;
(b2) 상기 수득한 융합단백질을 수세하는 단계; 및
(b3) 수세 후 균질화하는 단계로 이루어진 것인, 테리파라타이드의 생산방법.
The method of claim 1, wherein step (b)
(b1) obtaining a fusion protein in the form of an inclusion body from the cultured transformant, its culture, or its lysate;
(b2) washing the obtained fusion protein with water; And
(b3) The method of producing teriparatide, which consists of a step of homogenizing after washing with water.
The method of claim 1, wherein the TEV protease is contained in an amount of 0.01 to 0.1% by weight relative to the fusion protein, and is treated at 20 to 40°C for 3 to 6 hours.
The method of claim 5, wherein the TEV protease is contained in an amount of 0.06 to 0.07% by weight relative to the fusion protein, and is treated at 30° C. for 4 to 5 hours, teriparatide mass production method.
The method of claim 1, wherein a precipitation method is further performed after the adsorption chromatography and before treatment with TEV protease.
Teriparatide fusion protein encoded by the nucleotide sequence of SEQ ID NO: 1.
A teriparatide fusion protein expression vector comprising the nucleotide sequence of SEQ ID NO: 1.
A transformant into which the expression vector of claim 9 has been introduced.
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