Citation: MU Rong-Zi, SUN Xuan, WU Geng. Expression and Purification of Proto-oncoprotein KRas and Its Molecular Docoking with Small Molecule Inhibitor ZCL1688[J]. Chinese Journal of Analytical Chemistry, ;2022, 50(2): 263-270. doi: 10.19756/j.issn.0253-3820.210554 shu

Expression and Purification of Proto-oncoprotein KRas and Its Molecular Docoking with Small Molecule Inhibitor ZCL1688

School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

Corresponding author: WU Geng, geng.wu@sjtu.edu.cn
Received Date: 8 June 2021
Revised Date: 16 December 2021

Fund Project: Supported by the National Natural Science Foundation of China (No.31670106)

Specially designed small molecule compounds can covalently bind to the catalytic domain of KRas, thereby inhibiting the effect of downstream effector molecules. In this work, a high-purity KRas protein catalytic domain (AA 1-169) was prepared by nickel column affinity chromatography, enzyme cleavage labeling, gel chromatography and other steps. The results showed that KRas protein bound to the small molecule inhibitor ZCL1688 through K42 residue by isothermal titration calorimetry (ITC) experiments. Next, the modeled structure of the complex of KRas and ZCL1688 was obtained through molecular docking, and it was found that the combination of the two molecules had a significant competitive effect on the binding of KRas to down stream protein, serine/threonine kinase RAF. A simple method for high-efficiency expression and high-purity preparation of the KRas protein catalytic domain (AA 1-169) were thus established. The binding mode and weak interaction between KRas and ZCL1688 were analyzed. It laid a good foundation for the subsequent screening of the binding conditions of the protein with small molecule inhibitors and analysis of complex structure.
- KRas,
- Serine/threonine kinase RAF,
- Small molecule inhibitor,
- Protein purification,
- Molecular docking,
- Weak interaction
1. [1]
  FAN G J, SONG Z D, ZHANG X L, XIONF X F. Eur. J. Med. Chem., 2021, 226: 113816.
2. [2]
  DHARMAIAH S, TRAN T H, MESSING S, AGAMASU C, GILLETTE W K, YAN W, WAYBRIGHT T, ALEXANDER P, ESPOSITO D, NISSLEY D V, MCCORMICK F, STEPHEN A G, SIMANSHU D K. Sci. Rep., 2019, 9: 10512.
3. [3]
  UPRETY D, ADJEI A A. Cancer Treat. Rev., 2020, 89(2): 1-14.
4. [4]
  SIMANSHU D K, NISSLEY D V, MCCORMICK F. Cell, 2017, 170(1): 17-50.
5. [5]
  PAPKE B, DER C J. Science, 2017, 355(17): 1158-1163.
6. [6]
  HAIGIS K M. Trends Cancer, 2017, 3(10): 686-698.
7. [7]
  HAYAMA T, HASHIGUCHI Y, OKAMOTO K, OKADA Y, ONO K, SHIMADA R, OZAWA T, TOYODA T, TSUCHIYA T, IINUMA H, NOZAWA K, MATSUDA K. Int. J. Colorectal Dis., 2019, 34(8): 1491-1496.
8. [8]
  OSBORNE J K, MINNA J D. Cell Stem Cell, 2020, 27(4): 499-500.
9. [9]
  FU X H, CHEN Z T, WANG W H, FAN X J, HUANG Y, WU X B, HUANG J L, WANG J X, LIN H J,TAN X L, WANG L, WANG J P. J. Cancer, 2019, 10(4): 821-828.
10. [10]
  KIM D, XUE J Y, LITO P. Cell, 2020, 183(4): 850-859.
11. [11]
  OWEN D H, KONDA B, SIPOS J, LIU T, WEBB A, RINGEL M D, TIMMERS C D, SHAH M H. J. Natl. Compr. Cancer Network, 2019, 17(5): 409-413.
12. [12]
  OSTREM J M, PETERS U, SOS M L, WELLS J A, SHOKAT K M. Nature, 2013, 503(7477): 548-551.
13. [13]
  MCCORMICK F. Clin. Cancer Res., 2015, 21(8): 1797-1801.
14. [14]
  LIU P, WANG Y, LI X. Acta Pharm. Sin. B, 2019, 9(5): 871-879.
15. [15]
  MADHYASTHA N, SAMANTHA S K, DITTAKAVI S, MARKOSE M, MALLURWAR S R, ZAINUDDIN M, MULLANGI R. Biomed. Chromatogr., 2020, 35(4): e5043-e5069.
16. [16]
  CANON J, REX K, SAIKI A Y, MOHR C, COOKE K, BAGAL D, GAIDA K, HOLT T, KNUTSON C G, KOPPADA N, LANMAN B A, WERNER J, RAPAPORT A S, SAN MIGUE T, ORTIZ R, OSGOOD T, SUN J R, ZHU X, MCCARTER J D, VOLAK L P, HOUK B E, FAKIH M G, O'NEI B H, PRICE T J, FALCHOOK G S, DESAI J, KUO J, GOVINDAN R, HONG D S, OUYANG W, HENARY H, ARVEDSON T, CEE V J, LIPFORD J R. Nature, 2019, 575(7781): 217-223.
17. [17]
  FELL J B, FISCHER J P, BAER B R, BLAKE J F, BOUHANA K, BRIERE D M, BROWN K D, BURGESS L E, BURNS A C, BURKARD M R, CHIANG H, CHICARELLI M J, COOK A W, GAUDINO J J, HALLIN J, HANSON L, HARTLEY D P, HICKEN E J, HINGORANI G P, HINKLIN R J, MEJIA M J, OLSON P, OTTEN J N, RHODES S P, RODRIGUEZ M E, SAVECHENKOV P, SMITH D J, SUDHAKAR N, SULLIVAN F X, TANG T P, VIGERS G P, WOLLENBERG L, CHRISTENSEN J G, MARX M A. J. Med. Chem., 2020, 63(13): 6679-6693.
18. [18]
  MISALE S, FATHERREE J P, CORTEZ E, LI C, BILTON S, TIMONINA D, MYERS D T, LEE D, GOMEZ-CARABALLO M, GREENBERG M, NANGIA V, GRENINGER P, EGAN R K, MCCLANAGHAN J, STEIN G T, MURCHIE E, ZARRINKAR P P, JANES M R, LI L S, LIU Y, HATA A N, BENES C H. Clin. Cancer Res., 2019, 25(2): 796-807.
19. [19]
  JANES M R, ZHANG J, LI L S, HANSEN R, PETERS U, GUO X, CHEN Y, BABBAR A, FIRDAUS S J, DARJANIA L, FENG J, CHEN J H, LI S, LI S, LONG Y O, THACH C, LIU Y, ZARIEH A, ELY T, KUCHARSKI J M, KESSLER L V, WU T, YU K, WANG Y, YAO Y, DENG X, ZARRINKAR P P, BREHMER D, DHANAK D, LORENZI M V, HU-LOWE D, PATRICELLI M P, REN P, LIU Y. Cell, 2018, 172(3): 578-589.
20. [20]
  PATRICELLI M P, JANES M R, LI L S, HANSEN R, PETERS U, KESSLER L V, CHEN Y, KUCHARSKI J M, FENG J, ELY T, CHEN J H, FIRDAUS S J, BABBAR A, REN P, LIU Y. Cancer Discovery, 2016, 6(3): 316-330.
21. [21]
  LU T. Molclus Program, Version x.x ed., Available from: http://www.keinsci.com/research/molclus.html.
22. [22]
  BANNWARTH C, CALDEWEYHER E, EHLERT S, HANSEN A, PRACHT P, SEIBERT J, SPICHER S, GRIMMES. Wiley Interdiscip. Rev.: Comput. Mol. Sci., 2020, 11(2): e01493.
23. [23]
  FRISCH M J, TRUCKS G W, SCHLEGEL H B, SCUSERIA G E, ROBB M A, CHEESEMAN J R, SCALMANI G, BARONE V, MENNUCCI B, PETERSSON G A, NAKATSUJI H, CARICATO M, LI X, HRATCHIAN H P, IZMAYLOV A F, BLOINO J, ZHENG G, SONNENBERG J L, HADA M, EHARA M, TOYOTA K, FUKUDA R, HASEGAWA J, ISHIDA M, NAKAJIMA T, HONDA Y, KITAO O, NAKAI H, VREVEN T, MONTGOMERY J A, PERALTA J E, OGLIARO F, BEARPARK M, HEYD J J, BROTHERS E, KUDIN K N, STAROVEROV V N, KEITH T, KOBAYASHI R, NORMAND J, RAGHAVACHARI K, RENDELL A, BURANT J C, IYENGAR S S, TOMASI J, COSSI M, REGA N, MILLAM J M, KLENE M, KNOX J E, CROSS J B, BAKKEN V, ADAMO C, JARAMILLO J, GOMPERTS R, STRATMANN R E, YAZYEV O, AUSTIN A J, CAMMI R, POMELLI C, OCHTERSKI J W, MARTIN R L, MOROKUMA K, ZAKRZEWSKI V G, VOTH G A, SALVADOR P, DANNENBERG J J, DAPPRICH S, DANIELS A B, FARKAS O, FORESMAN J, ORTIZ J V, CIOSLOWSKI J, FOX D J. Gaussian 09. Revision D.01 ed., 2013.
24. [24]
  GRIMME S. J. Comput. Chem., 2006, 27(15): 1787-1799.
25. [25]
  LEAVER-FAY A, TYKA M, LEWIS S M, LANGE O F, THOMPSON J, JACAK R, KAUFMAN K, RENFREW P D, SMITH C A, SHEFFLER W, DAVIS I W, COOPER S, TREUILLE A, MANDELL D J, RICHTER F, BAN Y E, FLEISHMAN S J, CORN J E, KIM D E, LYSKOV S, BERRONDO M, MENTZER S, POPOVIC Z, HAVRANEK J J, KARANICOLAS J, DAS R, MEILER J, KORTEMME T, GRAY J J, KUHLMAN B, BAKER D, BRADLEY P. Methods Enzymol., 2011, 487: 545-574.
26. [26]
  LI S C, NG Y K. Bioinformatics, 2010, 11(25): 1-12.
27. [27]
  LU T, CHEN F. J. Comput. Chem., 2012, 33(5): 580-592.
28. [28]
  TRAN T H, SIMANSHU D K. Available from: https://www1.rcsb.org/structure/6VJJ, 2020.
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