Time- and cost-efficient bacterial expression and purification of potato apyrase
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作者:
Karim, Jordan A.
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机构:
Univ Oklahoma, Dept Biochem & Mol Biol, Hlth Sci Ctr, Oklahoma City, OK 73104 USAUniv Oklahoma, Dept Biochem & Mol Biol, Hlth Sci Ctr, Oklahoma City, OK 73104 USA
Karim, Jordan A.
[1
]
Lambert, Nevin A.
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Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USAUniv Oklahoma, Dept Biochem & Mol Biol, Hlth Sci Ctr, Oklahoma City, OK 73104 USA
Lambert, Nevin A.
[2
]
Pioszak, Augen A.
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Univ Oklahoma, Dept Biochem & Mol Biol, Hlth Sci Ctr, Oklahoma City, OK 73104 USAUniv Oklahoma, Dept Biochem & Mol Biol, Hlth Sci Ctr, Oklahoma City, OK 73104 USA
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
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.
机构:
Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
Jang, Wonjo
;
Adams, C. Elizabeth
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Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
Adams, C. Elizabeth
;
Liu, Heng
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机构:
Univ Pittsburgh, Sch Med, Dept Pharmacol & Chem Biol, Pittsburgh, PA 15261 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
Liu, Heng
;
Zhang, Cheng
论文数: 0引用数: 0
h-index: 0
机构:
Univ Pittsburgh, Sch Med, Dept Pharmacol & Chem Biol, Pittsburgh, PA 15261 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
Zhang, Cheng
;
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Levy, Finn Olav
;
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Andressen, Kjetil Wessel
;
Lambert, Nevin A.
论文数: 0引用数: 0
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机构:
Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Charite, Inst Neurophysiol, D-10117 Berlin, GermanyUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Madry, Christian
;
Arancibia-Carcamo, I. Lorena
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UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Arancibia-Carcamo, I. Lorena
;
Kyrargyri, Vasiliki
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UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Kyrargyri, Vasiliki
;
Chan, Victor T. T.
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机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Chan, Victor T. T.
;
Hamilton, Nicola B.
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机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Kings Coll London, Wolfson Ctr Age Related Dis, London SE1 1UL, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Hamilton, Nicola B.
;
Attwell, David
论文数: 0引用数: 0
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机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
机构:
Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
Jang, Wonjo
;
Adams, C. Elizabeth
论文数: 0引用数: 0
h-index: 0
机构:
Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
Adams, C. Elizabeth
;
Liu, Heng
论文数: 0引用数: 0
h-index: 0
机构:
Univ Pittsburgh, Sch Med, Dept Pharmacol & Chem Biol, Pittsburgh, PA 15261 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
Liu, Heng
;
Zhang, Cheng
论文数: 0引用数: 0
h-index: 0
机构:
Univ Pittsburgh, Sch Med, Dept Pharmacol & Chem Biol, Pittsburgh, PA 15261 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
Zhang, Cheng
;
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机构:
Levy, Finn Olav
;
论文数: 引用数:
h-index:
机构:
Andressen, Kjetil Wessel
;
Lambert, Nevin A.
论文数: 0引用数: 0
h-index: 0
机构:
Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USAAugusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA
机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Charite, Inst Neurophysiol, D-10117 Berlin, GermanyUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Madry, Christian
;
Arancibia-Carcamo, I. Lorena
论文数: 0引用数: 0
h-index: 0
机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Arancibia-Carcamo, I. Lorena
;
Kyrargyri, Vasiliki
论文数: 0引用数: 0
h-index: 0
机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Kyrargyri, Vasiliki
;
Chan, Victor T. T.
论文数: 0引用数: 0
h-index: 0
机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Chan, Victor T. T.
;
Hamilton, Nicola B.
论文数: 0引用数: 0
h-index: 0
机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Kings Coll London, Wolfson Ctr Age Related Dis, London SE1 1UL, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
Hamilton, Nicola B.
;
Attwell, David
论文数: 0引用数: 0
h-index: 0
机构:
UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, EnglandUCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England