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DANIEL MARKOVICH, ANDREAS WERNER AND HEINI MURER
(1000 colonies) show at least a 1.5-3-fold transport activity over background; progressive subdivision of one positive master plate down to plates
of 100-200 produce a 5-8-fold increase in transport rate; subdivision down
to plates of 10-30 colanies show transport signals of more than 10-fold over
background; and finally down to 1 positive colony, Stimulation up to 40-fold
over background. It is most important, that with each subdivision of colanies, an increase in the stimulated transport rate is necessary before proceeding further. Forageneral overview used for expression cloning of transport proteins, see Figure 1.
Applications
Examples of expression cloning
Since the discovery of Xenopus laevis oocytes as an expression system, numerous laboratories, including our own, have applied this technology towards the cloning of membrane proteins. Below, we wish to list a few transport proteins that were isolated using the Xenopus oocyte expression cloning system: SGL Tl, coding for an intestinal Na+ /glucose cotransport protein
(Hediger et al, 1987); N aPi -1, encoding Na+ /phosphate cotransport (W erner
et al. 1991); rBAT-1 and rBAT-2, encoding dibasic and neutral amino acid
transport (Bertran et al. 1992; Wells and Hediger, 1992; Markovich et al.
1993b); NaPi-2 and NaPi-3, encoding Na+/phosphate cotransport (Magagnin et al. 1993); NaSi-1, encoding Na+/sulfate cotransport (Markovich et.
al.,1993a); Na+/bile acid (taurocholate) transport (Hagenbuch et al.
1992); sat-1, Na-independent sulfate transport (Bissig et al. 1993); GAT1, encoding a transporter for the neurotransmitter gamma-aminobutyric
acid (GABA) protein (Guastella et al. 1990); EAAC1, encoding a glutamate
transporter (Kanai and Hediger, 1992).
Despite some difficulties which may be encountered initially with protein expression ( depending on the substratetype and assay conditions ), this
method seems to be reliable in the isolation of functional proteins from all
origins. lt is currently being introduced and widely used in many laboratories. Expressioncloning in Xenopus oocytes has proven extensively to be a
rewarding technique for the isolation of novel (functional) proteins.
DANIEL MARKOVICH, ANDREAS WERNER AND HEINI MURER
(1000 colonies) show at least a 1.5-3-fold transport activity over background; progressive subdivision of one positive master plate down to plates
of 100-200 produce a 5-8-fold increase in transport rate; subdivision down
to plates of 10-30 colanies show transport signals of more than 10-fold over
background; and finally down to 1 positive colony, Stimulation up to 40-fold
over background. It is most important, that with each subdivision of colanies, an increase in the stimulated transport rate is necessary before proceeding further. Forageneral overview used for expression cloning of transport proteins, see Figure 1.
Applications
Examples of expression cloning
Since the discovery of Xenopus laevis oocytes as an expression system, numerous laboratories, including our own, have applied this technology towards the cloning of membrane proteins. Below, we wish to list a few transport proteins that were isolated using the Xenopus oocyte expression cloning system: SGL Tl, coding for an intestinal Na+ /glucose cotransport protein
(Hediger et al, 1987); N aPi -1, encoding Na+ /phosphate cotransport (W erner
et al. 1991); rBAT-1 and rBAT-2, encoding dibasic and neutral amino acid
transport (Bertran et al. 1992; Wells and Hediger, 1992; Markovich et al.
1993b); NaPi-2 and NaPi-3, encoding Na+/phosphate cotransport (Magagnin et al. 1993); NaSi-1, encoding Na+/sulfate cotransport (Markovich et.
al.,1993a); Na+/bile acid (taurocholate) transport (Hagenbuch et al.
1992); sat-1, Na-independent sulfate transport (Bissig et al. 1993); GAT1, encoding a transporter for the neurotransmitter gamma-aminobutyric
acid (GABA) protein (Guastella et al. 1990); EAAC1, encoding a glutamate
transporter (Kanai and Hediger, 1992).
Despite some difficulties which may be encountered initially with protein expression ( depending on the substratetype and assay conditions ), this
method seems to be reliable in the isolation of functional proteins from all
origins. lt is currently being introduced and widely used in many laboratories. Expressioncloning in Xenopus oocytes has proven extensively to be a
rewarding technique for the isolation of novel (functional) proteins.