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Sequence-Validated and Expression-Tested Human cDNA in a Dual Expression,,,Vector

fy mammalian expression of a protein in CHO cells of the predicted molecular weight (Figure 4).

Fig.4

Inducible High-Level Bacterial Expression

The GeneConnection expression-tested clones contain features designed for inducible high-level protein expression in bacterial cells. The vector contains the hybrid T7/lac O promoter and lac repressor gene (lac I) to regulate protein expression. Therefore, expression is inducible using isopropyl-1-thio-b-D-galatopyranoside (IPTG) in BL21(DE3) bacterial cells that contain the T7 RNA polymerase.

HIS6-Tagged Fusion Protein in Bacterial Cell Lysates

To demonstrate the inducible expression in bacterial cells, we expressed a fusion protein consisting of wild-type green fluorescent protein (GFP),7 c-myc, and HIS6 in BL21-Gold cells. GFP was chosen because it is easily detected under long wavelength UV in induced plate and liquid cultures and requires the formation of a homodimer for biological activity. Results of the enzymatic GFP assays demonstrate that GFP tagged with c-myc and HIS6 was biologically active; therefore, the presence of the tags did not affect its activity (data not shown). Biological activity was not detected in cells transformed with the pDual GC vector without an insert.

Fusion proteins consisting of the protein encoded by the cDNA insert, c-myc, and HIS6 expressed in bacteria are quickly and easily purified from bacterial cell lysates. To demonstrate this, we purified the wild-type GFP fusion protein using Ni-NTA resin (Qiagen) under native conditions. The fusion protein also contains a thrombin cleavage site between GFP and the c-myc epitope. Following purification, the c-myc epitope and HIS6 purification tags were separated from GFP by incubation with thrombin
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