Large-scale purification of functional recombinant human aquaporin-2.
The homotetrameric aquaporin-2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his-tagged human AQP2 (HT-AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT-AQP2 could be obtained per liter of bioreactor culture. HT-AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93+/-0.03x10(-13) cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large-scale expression of functional recombinant human AQP2 that is suitable for structural studies.
Werten
P J
PJ
Department of Cell Physiology, University of Nijmegen, The Netherlands.
Hasler
L
L
Koenderink
J B
JB
Klaassen
C H
CH
de Grip
W J
WJ
Engel
A
A
Deen
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000737:chemistry* / Q000302:isolation & purification*; D016367:Baculoviridae / Q000378:metabolism; D001671:Biochemistry / Q000379:methods; D019149:Bioreactors; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007313:Insecta; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
C496023:AQP2 protein, human; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006639:Histidine; D014508:Urea