The M4M5 cytoplasmic loop of the Na,K-ATPase, overexpressed in Escherichia coli, binds nucleoside triphosphates with the same selectivity as the intact native protein.

The Journal of Biological Chemistry
Craig GattoJ H Kaplan

Abstract

Escherichia coli was used to overexpress the large cytoplasmic loop of the rat Na,K-ATPase. A 1260-base DNA segment encoding Lys354-Lys774 of the rat alpha1-subunit was constructed via polymerase chain reaction. The polymerase chain reaction product was successfully subcloned into the expression vector pET-28 (Novagen), which produces an N-terminal 6-histidine-tagged fusion protein. The pET-28 vector containing rat alpha-loop, i.e. pAN, was used to transform calcium-competent E. coli BL21(DE3) cells, and positive clones were selected by kanamycin resistance. Bacterial cultures were grown, and protein synthesis was induced with isopropyl beta-D-thiogalactoside. Cells were harvested and lysed, revealing production of the His-tagged fusion protein ( approximately 46 kDa). The fusion protein was affinity-purified from other soluble cellular proteins via a Ni-NTA column, which routinely yielded approximately 20 mg of soluble His6-alpha-loop/L cell culture. The His6-alpha-loop retained significant native structure, as evidenced by the ability of ATP and ADP (but not AMP, CTP, GTP, or UTP) to protect against chemical modification by either fluorescein isothiocyanate or maleimidylanilinonapthalene sulfonic acid. More specifically, circ...Continue Reading

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Citations

Aug 27, 2013·Plant Physiology and Biochemistry : PPB·Charitha GalvaCraig Gatto
May 31, 2007·Chemical Society Reviews·Xiangyang LiangPeter J Sadler
Jun 26, 2009·The Journal of Biological Chemistry·Rebecca J Clifford, Jack H Kaplan
Jun 15, 2012·The Journal of Biological Chemistry·Elmira TokhtaevaOlga Vagin
Sep 18, 2004·European Journal of Biochemistry·Rita KrumscheidWilhelm Schoner
Jun 5, 2002·Annual Review of Biochemistry·Jack H Kaplan
Jan 14, 2003·Annual Review of Physiology·Peter L JorgensenSteven J D Karlish
Aug 15, 2008·American Journal of Physiology. Renal Physiology·Rebecca J Clifford, Jack H Kaplan
May 24, 2003·Annals of the New York Academy of Sciences·Philipp PagelMichael J Caplan
Feb 24, 2009·Biochimica Et Biophysica Acta·Lenka GrycovaMartin Kubala
Sep 11, 2002·Biochemical and Biophysical Research Communications·Martin KubalaEvzen Amler
Nov 13, 2012·Biochimica Et Biophysica Acta·Marika HavlíkováMartin Kubala
Mar 27, 2018·Journal of Enzyme Inhibition and Medicinal Chemistry·Jaroslava ŠeflováMartin Kubala
Jun 23, 2004·The Journal of Biological Chemistry·Melissa LaugheryJack H Kaplan
May 20, 2003·FEBS Letters·Marc BramkampKarlheinz Altendorf
Apr 13, 2000·FEBS Letters·H OgawaC Toyoshima
Feb 24, 2006·The Journal of Biological Chemistry·Matthew H SazinskyAmy C Rosenzweig
Jun 6, 2006·The Journal of Biological Chemistry·A Brett MasonCarolyn W Slayman
Aug 15, 2001·American Journal of Physiology. Cell Physiology·C GattoJ H Kaplan
Aug 2, 2001·Clinical and Experimental Hypertension : CHE·D Salgado-CommissariatC H Pedemonte
Jan 4, 2003·The Journal of Biological Chemistry·Charles J CostaJack H Kaplan
Sep 24, 2004·Biochemistry·Marc Bramkamp, Karlheinz Altendorf
Jul 17, 2007·Biochemistry·Natalya U Fedosova, Mikael Esmann
Jun 7, 2008·Archives of Biochemistry and Biophysics·Svetlana LutsenkoVesna Zuzel
May 28, 2003·Biochemistry·Martin KubalaEvzen Amler

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