| Most of the commonly used enzyme probes are obtained by chemical conjugation of a signal protein with a binding protein. The use of chemical conjugation usually results in the presence of unreacted components leading to high background on the solid phases as well as variability between different preparations of the probe.The aim of this study is development of a genetically fused probe comprising alkaline phosphatase as a signal protein and a binding protein such as streptavidin. The fusion probe is thus advantageous over the chemically conjugated probe because of its compositional purity that significantly enhances the signal/background ratio and therefore the assay sensitivity while offering clonability and uniformity of the probe preparations. Such fusion probe would be of enormous commercial implication as a replacement to the traditional chemical probe.We have chosen alkaline phosphatase from E coli MG1655and monomeric streptavidin from Bacillus subtilis as fusion partners. Using site-directed mutagenesis, we constructed pET22a (+)-EAP expression system with Asp101â†'Ser mutation. Using gene synthesis method, we constructed pET-24a (+)-SA expression system with T76R, V125R, V55T, and the L109T four mutations. Finally, using standard PCR-restriction cloning technique, we constructed pET-24a (+)-SA-EAP and pET-24a (+)-EAP-SA expression systems.The results show that the mutant alkaline phosphatase was expressed with a higher level of soluble fraction and an enzymatic activity8.5-fold higher than the wild-type. The mutant streptavidin expressed at22℃existed in cell-free extract at a concentration of about0.8mg/mL. In contrast to its natural tetrameric wild-type, the mutant streptavidin specifically reacted with HABA with a change of the dye's absorption maximum to460nm. Expression of the SA-EAP and EAP-SA fusion proteins led to a low soluble content and a high level of inclusion bodies, necessitating further improvement. |
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