A fusion protein expression analysis using surface plasmon resonance imaging
Section snippets
Construction of plasmids
A gene encoding human growth hormone was amplified using a polymerase chain reaction (PCR) from a human cDNA library. The 3′ terminus was designed to contain a SalI restriction enzyme cleavage site. The PCR product was purified using a DNA purification kit (Qiagen) and digested with the restriction enzyme SalI. To introduce hexahistidine and ubiquitin tags at the N-terminus of the human growth hormone (His6-Ub-hGH), a gene encoding ubiquitin was amplified using the 5′ primer (CAT ATG CAC CAC
Results and discussion
The hexahistidine-ubiquitin-tagged hGH (His6-Ub-hGH), which was expressed in a soluble form, was partially purified using a one-step metal affinity chromatography, and analyzed using both SPR imaging and SDS–PAGE (Fig. 2). The SPR images were captured at an incident angle that was lower than the SPR angle of the background surface. The brighter spots are indications of the affinity binding of the target proteins on the IDA-Ni(II) chip. Protein microarray images with varying protein
Acknowledgements
This research was supported by a grant from the KRIBB Initiative Research Program. This work was also supported in part by a grant from KOSEF through the Center for Advanced Bioseparation Technology (BSEP).
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