The ability to purify an intact, functional protein or protein complex is an essential step in biochemical characterization studies. Challenges in purification arise when proteins are of low ...abundance or are unstable resulting in low yields or poor in vitro activity. In this protocol we describe a method to purify active, recombinant human proteins fused to a tandem MBP tag after expression in human 293T cells.
The goal of this study was to develop a mammalian expression system for the cloned rat intestinal, Na(+)-dependent, purine-selective nucleoside transporter (SPNTint) and to study the interactions of ...nucleosides and nucleoside analogs with this transporter.
Lipofection was used to transfect HeLa cells with a mammalian expression vector (pcDNA3) containing the cDNA insert encoding SPNTint. Nucleoside transport activity was measured using 3Hinosine, 3Huridine, 3H-dideoxyinosine (ddI), and 3H-2-chloro-2'-deoxyadenosine (2CdA) as model substrates.
Expression of SPNTint was observed between 36 and 90 h post-transfection, with maximal expression at 66 h. At 66 h, Na(+)-stimulated uptake of 3Hinosine in cells transiently transfected with SPNTint was approximately threefold greater than that in cells transfected with empty vector (p < 0.05). The Na(+)-stimulated uptake of both inosine and uridine was saturable (K(m) = 28.1 +/- 7.1 microM and 20.6 +/- 5.6 microM, respectively) in the transfected cells and was significantly inhibited by the naturally occurring nucleosides (1 mM) inosine and uridine and to a lesser extent by thymidine. The nucleoside analogs ddI (IC50 = 46 microM) and 2CdA (IC50 = 13 microM) also significantly inhibited the Na(+)-stimulated uptake of 3Hinosine. A Na(+)-stimulated uptake of 3H2CdA was observed suggesting that 2CdA is also a permeant of SPNTint.
HeLa cells transiently transfected with SPNTint represent a useful tool to study the kinetics and interactions of drugs with SPNTint.
Transgenic mice were developed that secreted chimeric mouse/human anti-human interleukin-2 receptor (IL-2R) antibodies (Ab) into their serum. In addition, hybridomas producing the chimeric Ab in ...tissue culture were generated from the transgenic mice. The presence of the mouse/human immunoglobulin (Ig) transgene did not appear to affect rearrangement of endogenous murine Ig in the hybridomas. Serum levels of the chimeric Ab correlated with transgene copy number. Although many of the transgenic lineages had serum titers of the chimeric Ab comparable to endogenous mouse IgG, there was no apparent correlation with endogenous mouse IgG levels.
