Time- and cost-efficient bacterial expression and purification of potato apyrase

被引:0
作者
Karim, Jordan A. [1 ]
Lambert, Nevin A. [2 ]
Pioszak, Augen A. [1 ]
机构
[1] Univ Oklahoma, Dept Biochem & Mol Biol, Hlth Sci Ctr, Oklahoma City, OK 73104 USA
[2] Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
基金
美国国家卫生研究院;
关键词
Nucleotide phosphohydrolase; Recombinant protein expression; E; coli trxB gor; Cibacron blue dye affinity chromatography; GPCR-G protein coupling BRET assay; ATP-DIPHOSPHOHYDROLASE APYRASE; PROTEINS; CLONING;
D O I
10.1016/j.pep.2022.106215
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Apyrase from potato (Solanum tuberosum) is a divalent metal ion-dependent enzyme that catalyzes the hydrolysis of nucleoside di- and tri-phosphates with broad substrate specificity. The enzyme is widely used to manipulate nucleotide levels such as in the G protein-coupled receptor (GPCR) field where it is used to deplete guanine nucleotides to stabilize nucleotide-free ternary agonist-GPCR-G protein complexes. Potato apyrase is available commercially as the native enzyme purified from potatoes or as a recombinant protein, but these are prohibitively expensive for some research applications. Here, we report a relatively simple method for the bacterial production of soluble, active potato apyrase. Apyrase has several disulfide bonds, so we co-expressed the enzyme bearing a C-terminal (His)6 tag with the E. coli disulfide isomerase DsbC at low temperature (18 degrees C) in the oxidizing cytoplasm of E. coli Origami B (DE3). This allowed low level production of soluble apyrase. A two-step purification procedure involving Ni-affinity followed by Cibacron Blue-affinity chromatography yielded highly purified apyrase at a level of similar to 0.5 mg per L of bacterial culture. The purified enzyme was functional for ATP hydrolysis in an ATPase assay and for GTP/GDP hydrolysis in a GPCR-G protein coupling assay. This methodology enables the time- and cost-efficient production of recombinant apyrase for various research applications.
引用
收藏
页数:6
相关论文
共 24 条
[1]   Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm [J].
Bessette, PH ;
Åslund, F ;
Beckwith, J ;
Georgiou, G .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (24) :13703-13708
[2]   ATP mediates rapid microglial response to local brain injury in vivo [J].
Davalos, D ;
Grutzendler, J ;
Yang, G ;
Kim, JV ;
Zuo, Y ;
Jung, S ;
Littman, DR ;
Dustin, ML ;
Gan, WB .
NATURE NEUROSCIENCE, 2005, 8 (06) :752-758
[3]   Cloning, expression, and characterization of salivary apyrase from Aedes albopictus [J].
Dong, Fang ;
Fu, Yongfeng ;
Li, Xueping ;
Jiang, Jianguo ;
Sun, Jianhua ;
Cheng, Xunjia .
PARASITOLOGY RESEARCH, 2012, 110 (02) :931-937
[4]   Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum) [J].
Handa, M ;
Guidotti, G .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1996, 218 (03) :916-923
[5]   An inactive receptor-G protein complex maintains the dynamic range of agonist-induced signaling [J].
Jang, Wonjo ;
Adams, C. Elizabeth ;
Liu, Heng ;
Zhang, Cheng ;
Levy, Finn Olav ;
Andressen, Kjetil Wessel ;
Lambert, Nevin A. .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2020, 117 (48) :30755-30762
[6]   Highly accurate protein structure prediction with AlphaFold [J].
Jumper, John ;
Evans, Richard ;
Pritzel, Alexander ;
Green, Tim ;
Figurnov, Michael ;
Ronneberger, Olaf ;
Tunyasuvunakool, Kathryn ;
Bates, Russ ;
Zidek, Augustin ;
Potapenko, Anna ;
Bridgland, Alex ;
Meyer, Clemens ;
Kohl, Simon A. A. ;
Ballard, Andrew J. ;
Cowie, Andrew ;
Romera-Paredes, Bernardino ;
Nikolov, Stanislav ;
Jain, Rishub ;
Adler, Jonas ;
Back, Trevor ;
Petersen, Stig ;
Reiman, David ;
Clancy, Ellen ;
Zielinski, Michal ;
Steinegger, Martin ;
Pacholska, Michalina ;
Berghammer, Tamas ;
Bodenstein, Sebastian ;
Silver, David ;
Vinyals, Oriol ;
Senior, Andrew W. ;
Kavukcuoglu, Koray ;
Kohli, Pushmeet ;
Hassabis, Demis .
NATURE, 2021, 596 (7873) :583-+
[7]   PROPERTIES OF 2 APYRASES FROM SOLANUM-TUBEROSUM [J].
KETTLUN, AM ;
URIBE, L ;
CALVO, V ;
SILVA, S ;
RIVERA, J ;
MANCILLA, M ;
VALENZUELA, MA ;
TRAVERSOCORI, A .
PHYTOCHEMISTRY, 1982, 21 (03) :551-558
[8]   Effects of the ecto-ATPase apyrase on microglial ramification and surveillance reflect cell depolarization, not ATP depletion [J].
Madry, Christian ;
Arancibia-Carcamo, I. Lorena ;
Kyrargyri, Vasiliki ;
Chan, Victor T. T. ;
Hamilton, Nicola B. ;
Attwell, David .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2018, 115 (07) :E1608-E1617
[9]  
Mirdita Milot, 2021, Zenodo
[10]   Reconstitution of R-Spondin:LGR4:ZNRF3 Adult Stem Cell Growth Factor Signaling Complexes with Recombinant Proteins Produced in Escherichia coli [J].
Moad, Heather E. ;
Pioszak, Augen A. .
BIOCHEMISTRY, 2013, 52 (41) :7295-7304