Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity.
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.
Chloupková
Maja
M
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430-6540, USA.
Pickert
Amanda
A
Lee
Jyh-Yeuan
JY
Souza
Shiloe
S
Trinh
Yenphuong T
YT
Connelly
Sara M
SM
Dumont
Mark E
ME
Dean
Michael
M
Urbatsch
Ina L
IL
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D010843:Pichia; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; C114462:multidrug resistance-associated protein 3