Elsevier

Analytical Biochemistry

Volume 330, Issue 2, 15 July 2004, Pages 251-256
Analytical Biochemistry

A fusion protein expression analysis using surface plasmon resonance imaging

https://doi.org/10.1016/j.ab.2004.02.009Get rights and content

Abstract

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli. With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His6-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blots.

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).

References (17)

There are more references available in the full text version of this article.

Cited by (47)

  • Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: A review

    2012, Biochemical Engineering Journal
    Citation Excerpt :

    The enumerated methods are time-consuming, labour-intensive, and destroy samples, making them poor techniques for optimizing protein production in most cases [64]. Advances in analytical technology have yielded improved protein monitoring methods, including perfusion chromatography [65–66], electrochemiluminescence [67], special biosensors [68–70] and immunonephlometric assays [71–72]. Baker et al. provide a detailed comparison of these methods [73].

View all citing articles on Scopus
View full text