Characterization of human taurine transporter expressed in insect cells using a recombinant baculovirus.
A recombinant baculovirus system was used to express the human taurine transporter in Sf9 cells and characterize its mediated uptake activity. This uptake process exhibited: (i) Na(+) dependence, (ii) larger inhibition of taurine transport by competing beta-amino acids than by alpha- and gamma-amino acids, (iii) apparent Michaelis constant, K(t), for taurine transport of 1.6 +/- 0.2 microM, and (iv) a maximal velocity, V(max), of 262 +/- 18 pmol/mg protein per 15 min. Coexpression of a molecular chaperone, human calnexin, enhanced taurine transporter activity by 43%. During development of taurine transporter expression, exposure to tunicamycin (10 microg/ml) decreased taurine transport activity by 76%. The taurine transporter linked to glutathione S-transferase (GST) was expressed to determine whether this conjugate also elicits taurine transport activity. Even though transport activity was markedly decreased, its Na(+) dependence was still evident. Coexpression of calnexin enhanced expression of this conjugated transporter activity by 54%. Immunoblot analysis revealed that calnexin did not change the amount of GST-taurine transporter conjugate or its molecular mass (i.e., 58.4-68.0 kDa). However, tunicamycin decreased its molecular mass. Taken together, taurine transport activity in a baculovirus expression system has characteristics similar to its wild-type counterpart. Stimulation of transport activity by coexpression with calnexin suggests the importance of transporter folding for optimal transport activity. Glycosylation of the transporter also increases its transport activity. Finally, GST-taurine transporter conjugate usage may aid transporter purification even though its transport activity decreases.
Miyasaka
T
T
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba, 277-8562, Japan.
Kaminogawa
S
S
Shimizu
M
M
Hisatsune
T
T
Reinach
P S
PS
Miyamoto
Y
Y
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001692:Biological Transport; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D026901:Membrane Transport Proteins; D017510:Protein Folding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics*; D013379:Substrate Specificity; D013654:Taurine / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology; D000099247:Taurine Transporters
D002135:Calcium-Binding Proteins; D037281:Calnexin; D002352:Carrier Proteins; D004279:DNA, Viral; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D012964:Sodium; D013654:Taurine; D014415:Tunicamycin; D000099247:Taurine Transporters