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