Functional expression of the human breast cancer resistance protein in Pichia pastoris

Biochemical and Biophysical Research Communications
Qingcheng MaoJashvant D Unadkat

Abstract

We report functional expression of BCRP in Pichia pastoris in which BCRP was produced as a 62 kDa underglycosylated protein. BCRP expression level in P. pastoris was comparable to that in HEK cells. The basal BCRP ATPase activity in the yeast membranes was approximately 40-80 nmol Pi/min/mg protein, which can be modulated by known BCRP substrates and inhibitors. Photolabeling of BCRP with 8-azido[alpha-32P]ATP was dependent preferentially on the presence of Co2+ than Mg2+ and could be inhibited by a molar excess of ATP. Vanadate-induced trapping of 8-azido[alpha-32P]ADP by BCRP was much more significant in the presence of Co2+ than that with Mg2+. The Km and Vmax values of BCRP for [3H]E1S transport were 3.6+/-0.3 microM and 55.2+/-1.6 pmol/min/mg protein, respectively. This efficient and cost-effective expression system should facilitate large scale production and purification of BCRP for further structural and functional analyses.

References

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Citations

Nov 9, 2007·Journal of Bioenergetics and Biomembranes·Saroj VelamakanniHendrik W van Veen
Mar 6, 2010·The Journal of Pharmacology and Experimental Therapeutics·Xiaokun CaiQingcheng Mao
Apr 16, 2009·Clinical Microbiology Reviews·Richard D CannonBrian C Monk
Sep 9, 2005·The AAPS Journal·Qingcheng Mao, Jashvant D Unadkat
Apr 10, 2015·Marine Drugs·Lilia CherigoSergio Martinez-Luis
Jan 7, 2009·Advanced Drug Delivery Reviews·Christopher A McDevittRichard Callaghan
Nov 4, 2008·Protein Expression and Purification·Alexandre PozzaAttilio Di Pietro
Mar 4, 2005·Drug Metabolism and Disposition : the Biological Fate of Chemicals·R Robert VethanayagamQingcheng Mao
Oct 25, 2017·Scientific Reports·Narakorn KhunweeraphongKarl Kuchler
Jul 9, 2009·Chemical Reviews·Paul D W Eckford, Frances J Sharom

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