[논문]재조합 Thymosin β-4의 세포이동능과 상처치유능

홍교창; 최영현; 김군도; 차희재; 전숭종; 남수완

doi:10.5352/jls.2022.32.2.135

재조합 Thymosin β-4의 세포이동능과 상처치유능
Cell Migration and Wound Healing Activities of Recombinant Thymosin β-4 Expressed in Escherichia coli 원문보기

생명과학회지 = Journal of life science, v.32 no.2, 2022년, pp.135 - 141

홍교창 (동의대학교 스마트바이오헬스학과) , 최영현 (동의대학교 스마트바이오헬스학과) , 김군도 (부경대학교 자연과학대학 미생물학과) , 차희재 (고신대학교 의과대학 기생충학 유전학 교실) , 전숭종 (동의대학교 스마트바이오헬스학과) , 남수완 (동의대학교 스마트바이오헬스학과)

초록
AI-Helper

Mouse thymosin β-4 (TB4) 유전자를 Escherichia coli에서 intein-tag 융합 단백질로 발현시키고 정제하였다. 재조합 TB4-intein 융합 단백질의 분자량을 10% glycine SDS-PAGE로 확인한 결과, 세포내 soluble 분획에서 60 kDa의 단백질을 얻을 수 있었고, 유도발현 최적 조건은 0.1 mM IPTG, 25℃에서 3시간 동안 유도 발현할 때가 최적임을 확인하였다. TB4 만을 얻기 위해서 유도발현된 TB4-intein을 DTT를 이용해 self-cleavage를 일으킨 다음, chitin bead를 이용한 친화성 크로마토그라피로 정제 후 분자량을 확인 한 결과, 5 kDa으로 확인되었으며, 순도는 95%이상 이었다. 정제한 recombinant TB4가 생물학적 기능을 보유하고 있는지 확인을 하기 위하여, TB4를 농도 별(1~1,000 ng/ml)로 하여 HT1080 cell을 이용한 cell migration을 측정한 결과, 모든 농도에서 recombinant TB4가 화학합성한 TB4 보다 약 20% 이상 높은 활성을 보였으며, recombinant TB4 1 ng/ml의 농도에서 cell migration 활성이 가장 높게 나타났다. Recombinant TB4를 마우스 상처 부위에 5일 동안 매일 처리한 결과(최종 처리 농도 0.5 mg/ml), 화학합성 TB4 보다 recombinant TB4의 상처치유 활성이 약 35% 더 높음을 알 수 있었다. 이상의 결과는 recombinant TB4가 화학합성 TB4보다 cell migration과 상처치유에 훨씬 높은 활성을 나타냄을 보여주고 있다.

Abstract ▼ AI-Helper

Thymosin β-4 (TB4) is a small peptide composed of 43 amino acids. To obtain sufficient biologically active mouse TB4 economically, we cloned and overexpressed this gene in an Escherichia coli system. With the isopropyl β-D-1-thiogalactopyranoside induction of the E. coli transformant, TB4 fusion protein with intein- and chitin-binding domain was successfully expressed in the soluble fraction within the E. coli cell. The TB4-intein - chitin-binding domain fusion protein was purified from the soluble fraction of E. coli cell lysate. The affinity chromatography with chitin beads and dithiothreitol-mediated intein self-cleavage reaction releases the TB4 peptide into the stripping solution. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis and Western blot analyses were used to confirm that the recombinant TB4 peptide was produced with the expected size of 5 kDa. We found that the recombinant TB4 stimulated cell migration in the transwell plate chamber assay. After 18 hr of the treatment of the recombinant TB4 with 1 ng/ml concentration, the migration of the HT1080 cell was increased by 20% compared with that of the chemically synthesized TB4. The recombinant TB4 was also observed to promote the healing of a wound area in C57BL/6 mice by as high as 35% compared with that of the chemically synthesized TB4. These results suggest that the recombinant TB4 has better biological activity for cell migration and wound healing than that of the chemically synthesized TB4 peptide.

주제어

표/그림 (4)

그림 Fig. 1. 10% Glycine SDS-PAGE analysis of the recombinant TB4- intein fusion protein. Lane 1, protein molecular weight markers; lane 2, cell lysate of E. coli BL21 (DE3) Codon plus; lane 3, cell lysate of E. coli BL21 (DE3) Codon plus/pTYB2-TB4 (37℃, 0.5 mM IPTG, 3 hr).
$Fig. 2. Purification of recombinant TB4 expressed in E. coli. (A) 10% glycine SDS-PAGE of E. coli BL21 (DE3) Codon plus/pTYB2-TB4. Lane 1, protein molecular weight markers; lane 2, crude extract of transformant E. coli cells; lane 3, supernatant of crude extract of transformant E. coli cells; lane 4, non-binding proteins to chitin beads; lane 5, elution fraction after cleavage with DTT; lane 6, intein-CBD fraction. (B) 16% Tricine SDS-PAGE of purified TB4. Lane 1, protein molecular weight markers; lane 2, purified TB4. (C) Western blotting of recombinant TB4.$ 그림 Fig. 2. Purification of recombinant TB4 expressed in E. coli. (A) 10% glycine SDS-PAGE of E. coli BL21 (DE3) Codon plus/pTYB2-TB4. Lane 1, protein molecular weight markers; lane 2, crude extract of transformant E. coli cells; lane 3, supernatant of crude extract of transformant E. coli cells; lane 4, non-binding proteins to chitin beads; lane 5, elution fraction after cleavage with DTT; lane 6, intein-CBD fraction. (B) 16% Tricine SDS-PAGE of purified TB4. Lane 1, protein molecular weight markers; lane 2, purified TB4. (C) Western blotting of recombinant TB4.
그림 Fig. 3. Staining of migrated HT1080 cell with hematoxylin. (A), control (PBS); (B). 1 ng/ml chemical TB4; (C), 1 ng/ml recombinant TB4. Scale bar 10 μm. (D), Effects of TB4 concentration on HT1080 cell migration. (black bar, chemical TB4; grey bar, recombinant TB4). Student's t-test, p<0.01.
그림 Fig. 4. Microphotos of wound tissues after treatment of TB4 for 5 days. (A), control (30% glycerol); (B), chemical TB4; (C), recombinant TB4; (D), Wound healing activity of recombinant TB4 (final concentration=0.5 mg/ml). Student’s t-test, p<0.01. (scale bar = 100 μm).

참고문헌 (31)

Belsky, J. B., Rivers, E. P., Filbin, M. R., Lee, P. J. and Morris, D. C. 2018. Thymosin beta 4 regulation of actin in sepsis. Expert Opin. Biol. Ther. 18, 193-197.

상세보기
Bock-Marquette, I., Saxena, A., White, M. D., DiMaio, J. M. and Srivastava, D. 2004. Thymosin β4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432, 466-472.

상세보기
Carpintero, P., Anadon, R., Diaz-Regueira, S. and GomezMarquez, J. 1999. Expression of thymosin β 4 messenger RNA in normal and kainate-treated rat forebrain. Neuroscience 90, 1433-1444.

상세보기
Cha, H. J., Jeong, M. J. and Kleinman, H. K. 2003. Role of thymosin β4 in tumor metastasis and angiogenesis. J. Natl. Cancer Inst. 95, 1674-1680.

상세보기
Dai, B., Sha, R. N., Yuan, J. L. and Liu, D. J. 2021. Multiple potential roles of thymosin β4 in the growth and development of hair follicles. J. Cell Mol. Med. 25, 1350-1358.

상세보기
Esipov, R. S., Makarov, D. A., Stepanenko, V. N. and Miroshnikov, A. I. 2016. Development of the intein-mediated method for production of recombinant thymosin β4 from the acetylated in vivo fusion protein. J. Biotechnol. 228, 73-81.

상세보기
Freeman, K. W., Bowman, B. R. and Zetter, B. R. 2011. Regenerative protein thymosin β-4 is a novel regulator of purinergic signaling. FASEB J. 25: 907-915.

상세보기
Gomez-Marquez J., Dosil, M., Segade, F., Bustelo, X. R., Pichel, J. G., Dominguez, F. and Freire, M. 1989. Thymosinbeta 4 gene, preliminary characterization and expression in tissues, thymic cells, and lymphocytes. J. Immunol. 143, 2740-2744.

상세보기
Kaur, H. and Mutus, B. 2012. Platelet function and thymosin β4. Biol. Chem. 393, 595-598.

상세보기
Kaveh-Baghbaderani, Y., Blank-Shim, S. A., Koch, T. and Berensmeier, S. 2018. Selective release of overexpressed recombinant proteins from E. coli cells facilitates one-step chromatographic purification of peptide-tagged green fluorescent protein variants. Protein Expr. Purif. 152, 155-160.

상세보기
Kim, S. H. and Kwon, J. K. 2014. Thymosin beta 4 improves dermal burn wound healing via downregulation of receptor of advanced glycation end products in db/db mice. Biochim. Biophys. Acta. 1840, 3452-3459.

상세보기
Kobayashi, T., Okada, F., Fujii, N., Tomita, N., Ito, S., Tazawa, H. and Hosokawa, M. 2002. Thymosin-β4 regulates motility and metastasis of malignant mouse fibrosarcoma cells. Am. J. Pathol. 160, 869-882.

상세보기
Kozaczuk, A., Selmi, A. and Bednarek, R. 2013. Bacterial expression, purification and angiogenesis-promoting activity of human thymosin β4. Protein Expr. Purif. 90, 142-152.

상세보기
Li, T., Ma, S. Y., Tang, X. C., Nie, L. Y. and Huang, H. 2013. Production and characterization of highly purified recombinant thymosin beta 4 in Escherichia coli. Protein Expr. Purif. 90, 90-95.

상세보기
Li, X., Zheng, L., Peng, F., Qi, C., Zhang, X., Zhou, A. and Wu, S. 2007. Recombinant thymosin beta 4 can promote full-thickness cutaneous wound healing. Protein Exp. Purif. 56, 229-236.

상세보기
Lv, S., Cai, H., Xu, Y., Dai, J., Rong, X. and Zheng, L. 2020. Thymosin-β 4 induces angiogenesis in critical limb ischemia mice via regulating Notch/NF-κB pathway. Int. J. Mol. Med. 46, 1347-1358.
Malinda, K. M., Goldstein, A. L. and Kleinman, H. K. 1997. Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells. FASEB J. 11, 474-481.

상세보기
Malinda, K. M., Sidhu, G. S., Mani, H., Banaudha, K., Maheshwari, R. K., Goldstein, A. L. and Kleinman, H. K. 1999. Thymosin β4 accelerates wound healing. J. Invest. Dermatol. 113, 364-368.

상세보기
Martinez, A., Knappskog, P. M., Olafsdottir, S., Doskeland, A. P., Eiken, H. G., Svebak, R. M., Bozzini, M., Apold, J. and Flatmark, T. 1995. Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme. Biochem. J. 306, 589-597.

상세보기
Otto, A., Muller, C., Huff, T. and Hannappel, E. 2002. Chemotherapeutic drugs change actin skeleton organization and the expression of β-thymosins in human breast cancer cells. J. Cancer Res. Clin. Oncol. 128, 247-256.

상세보기
Philp, D., Goldstein, A. L. and Kleinman, H. K. 2004. Thymosin β 4 promotes angiogenesis, wound healing, and hair follicle development. J. Coloproctol. 125, 113-115.
Qiu, P., Kurpakus-Wheater, M. and Sosne, G. 2007. Matrix metalloproteinase activity is necessary for thymosin beta 4 promotion of epithelial cell migration. J. Cell. Physiol. 212, 165-173.

상세보기
Renga, G., Oikonomou, V., Stincardini, C., Pariano, M., Borghi, M., Costantini, C., Bartoli, A., Garaci, E., Goldstein, A. L. and Romani, L. 2018. Thymosin β4 limits inflammation through autophagy. Expert Opin. Biol. Ther. 18, 171-175.

상세보기
Skosyrev, V. S., Kulesskiy, E. A., Yakhnin, A. V., Temirov, Y. V. and Vinokurov, L. M. 2003. Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells. Protein Expr. Purif. 28, 350-356.

상세보기
Sosne, G. 2018. Thymosin beta 4 and the eye: the journey from bench to bedside. Expert Opin. Biol. Ther. 18, 99-104.

상세보기
Stoeckli, M., Chaurand, P., Hallahan, D. E. and Caprioli, R. M. 2001. Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. Nat. Med. 7, 493-496.

상세보기
Tsuji, Y., Kitahara-Tanabe, N., Noguchi, K., Gatanaga, T., Mizuno, D. and Soma, G. 1989. Production in Escherichia coli of human thymosin beta 4 as chimeric protein with human tumor necrosis factor. Biochem. Int. 18, 501-508.

상세보기
Wang, X., Yang, G., Li, S., Gao, M., Zhao, P. and Zhao, L. 2013. The Escherichia coli-derived thymosin β4 concatemer promotes cell proliferation and healing wound in mice. Biomed. Res. Int. 2013, Article ID 241721.
Xu, T. J., Wang, Q., Ma, X. W., Zhang, Z., Zhang, W., Xue, X. C., Zhang, C., Hao, Q., Li, W. N., Zhang, Y. Q. and Li, M. 2013. A novel dimeric thymosin beta 4 with enhanced activities accelerates the rate of wound healing. Drug Des. Devel. Ther. 7, 1075-1088.

상세보기
Yu, R., Cao, S., Liu, Y., Si, X., Fang, T., Sun, X., Dai, H., Xu, J., Fang, H. and Chen, W. 2018. Highly effective biosynthesis of N-acetylated human thymosin β4 (Tβ4) in Escherichia coli. Artif. Cells Nanomed. Biotechnol. 46, S95-S104.

상세보기
Zhou, Q., Jiang, S., Ma, K. and Li, C. 2014. Expression of a novel recombinant fusion protein BVN-Tβ4 and its effects on diabetic wound healing. J. Biosci. Bioeng. 118, 341-343.

상세보기

저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증