Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity

Maja Chloupková, Amanda Pickert, Jyh Yeuan Lee, Shiloe Souza, Yenphuong T. Trinh, Sara M. Connelly, Mark E. Dumont, Michael Dean, Ina L. Urbatsch

Research output: Contribution to journalArticle

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Abstract

Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanning transport proteins, many of which are associated with genetic diseases or multidrug resistance of cancers. In this study, we present a comprehensive approach for the cloning, expression, and purification of human ABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstrate that 11 transporters, including ABCC3, ABCB6, ABCD1, ABCG1, ABCG4, ABCG5, ABCG8, ABCE1, ABCF1, ABCF2, and ABCF3, were expressed at high levels comparable to that of ABCB1 (P-glycoprotein). As an example of the purification strategy via tandem affinity chromatography, we purified ABCC3 (MRP3) whose role in the transport of anticancer drugs, bile acids, and glucuronides has been controversial. The yield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activated with PC lipids, exhibited significant ATPase activity with a Vmax of 82 ± 32 nmol min-1 mg-1. The ATPase activity was stimulated by bile acids and glucuronide conjugates, reaching 170 ± 28 nmol min-1 mg-1, but was not stimulated by a variety of anticancer drugs. The glucuronide conjugates ethinylestradiol-3-glucuronide and 17β-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities (apparent Km values of 2 and 3 μM, respectively) in contrast to bile acids (apparent Km values of > 130 μM), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availability of a purification system for the production of large quantities of active transporters presents a major step not only toward understanding the role of ABCC3 but also toward future structure-function analysis of other human ABC transporters.

Original languageEnglish (US)
Pages (from-to)7992-8003
Number of pages12
JournalBiochemistry
Volume46
Issue number27
DOIs
StatePublished - Jul 10 2007
Externally publishedYes

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ATP-Binding Cassette Transporters
Pichia
Glucuronides
Yeast
Adenosine Triphosphatases
Yeasts
Bile Acids and Salts
Purification
Inborn Genetic Diseases
Ethinyl Estradiol
Disease Resistance
Membrane Transport Proteins
Affinity chromatography
Multiple Drug Resistance
P-Glycoprotein
Affinity Chromatography
Pharmaceutical Preparations
Cloning
Organism Cloning
Estradiol

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chloupková, M., Pickert, A., Lee, J. Y., Souza, S., Trinh, Y. T., Connelly, S. M., ... Urbatsch, I. L. (2007). Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity. Biochemistry, 46(27), 7992-8003. https://doi.org/10.1021/bi700020m

Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity. / Chloupková, Maja; Pickert, Amanda; Lee, Jyh Yeuan; Souza, Shiloe; Trinh, Yenphuong T.; Connelly, Sara M.; Dumont, Mark E.; Dean, Michael; Urbatsch, Ina L.

In: Biochemistry, Vol. 46, No. 27, 10.07.2007, p. 7992-8003.

Research output: Contribution to journalArticle

Chloupková, M, Pickert, A, Lee, JY, Souza, S, Trinh, YT, Connelly, SM, Dumont, ME, Dean, M & Urbatsch, IL 2007, 'Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity', Biochemistry, vol. 46, no. 27, pp. 7992-8003. https://doi.org/10.1021/bi700020m
Chloupková, Maja ; Pickert, Amanda ; Lee, Jyh Yeuan ; Souza, Shiloe ; Trinh, Yenphuong T. ; Connelly, Sara M. ; Dumont, Mark E. ; Dean, Michael ; Urbatsch, Ina L. / Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity. In: Biochemistry. 2007 ; Vol. 46, No. 27. pp. 7992-8003.
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