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

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

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

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

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 17beta-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities (apparent Km values of 2 and 3 microM, respectively) in contrast to bile acids (apparent Km values of >130 microM), 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.

Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity. Publishing Authors By Initials

M ,A Pickert,JY Lee,S Souza,YT Trinh,SM Connelly,ME Dumont,M Dean,IL Urbatsch,

For similar proteins: recombinant proteins research abstracts see: proteins: recombinant proteins research

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

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Biochemistry

VOLUME: 46

Page Numbers: 7992-8003

Journal Abbreviation: Biochemistry

ISSN: 0006-2960

DAY: 15

MONTH: 06

YEAR: 2007

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

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LANGUAGE: eng

NlmUniqueID: 370623

Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity. Keywords Mesh Terms:

KEYWORDS: Recombinant Proteins

MESH TERMS: genetics

Chemical & Substance for Abstract: Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity. Information

Substance Name: Adenosine Triphosphate

Registry Number: 56-65-5

Grant and Affiliation Information for Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity.

AFFILIATION: Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430-6540, USA.

Country: United States

AGENCY: United States NIGMS

GRANT: P50GM64655

ACRONYM: GM

MEDLINETA: Biochemistry

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