Elsevier

Analytical Biochemistry

Volume 451, 15 April 2014, Pages 63-68
Analytical Biochemistry

A human kringle domain-based fluorescence-linked immunosorbent assay system

https://doi.org/10.1016/j.ab.2014.01.019Get rights and content

Abstract

As a non-immunoglobulin protein scaffold, human kringle domain (KD) has attractive properties such as high specificity, stability, and production in bacterial hosts. Here, we developed a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) system using a fluorescent kringle domain (fluoKD), a fusion protein of a green fluorescent protein (GFP), and a kringle domain variant (KD548). Two kinds of fluoKDs in which KD was fused to the N terminus of GFP (N-fluoKD) or the C terminus of GFP (C-fluoKD) were constructed and characterized. In Escherichia coli host, both fluoKDs were produced in high yield and solubility and were successfully purified by a simple procedure. The purified fluoKDs exhibited strong fluorescent activities and high affinities to the target antigen. Furthermore, it was successfully demonstrated that the FLISA with purified fluoKDs allowed for more rapid detection of target antigens with higher sensitivity compared with conventional enzyme-linked immunosorbent assay (ELISA), indicating that a simple, rapid, and sensitive immunoassay system could be developed by using KD instead of antibody or antibody fragments.

Section snippets

Bacterial strains and plasmid

All bacterial strains used in this work are listed in Table 1. E. coli XL1-Blue was used as a host cell for gene manipulation and plasmid maintenance. E. coli SHuffle Express, which is engineered to promote disulfide bond formation in the cytoplasm by constitutive expression of DsbC in cytoplasm, was used for production of both fluoKDs and GFP. Even though KD has three disulfide bonds, the correctly folded KD can be produced in cytoplasm of this strain. Polymerase chain reaction (PCR) was

Production and purification of fluoKDs

For the production of both fluoKDs (anti-DR5 KD548 fused GFPs), the constructed plasmids, pGM-N-fKD and pGM-C-fKD, were transformed into E. coli SHuffle Express for production. After flask cultivation, the production yields and solubility of each fluoKD were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In the cytoplasm of E. coli, both fluoKDs were highly expressed (N-fluoKD, 17% of total proteins; C-fluoKD, 8% of total proteins) with high solubility (>90%) (

Discussion

Since the last decade, there has been considerable progress in the development of non-immunoglobulin protein scaffolds [12], [13]. In addition to having similar properties as antibodies, protein scaffolds have several useful features such as small size (<100 amino acids), high thermostability, and economic production in bacterial hosts. Owing to these features, they have been considered as potential alternatives to antibodies for therapeutic and diagnostic purposes [5]. In many diagnostic

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