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The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than green light and being less absorbed by biological tissues, red-emitting luciferases have been considered as useful reporters in imaging technology. A set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase has been made by site-directed mutagenesis. Among different beetle luciferases, those from Phrixothrix (railroad worm) emit either green or red bioluminescence colours naturally. By substitution of three specific amino acids using site-specific mutagenesis in a green-emitting luciferase (from L. turkestanicus), the colour of emitted light was changed to red concomitant with decreasing decay rate. Different specific mutations (H245N, S284T and H431Y) led to changes in the bioluminescence colour. Meanwhile, the luciferase reaction took place with relative retention of its basic kinetic properties such as K(m) and relative activity. Structural comparison of the native and mutant luciferases using intrinsic fluorescence, far-UV CD spectra and homology modelling revealed a significant conformational change in mutant forms. A change in the colour of emitted light indicates the critical role of these conserved residues in bioluminescence colour determination among firefly luciferases. Relatively high specific activity and emission of red light might make these mutants suitable as reporters for the study of gene expression and bioluminescence imaging.

Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.

Renilla luciferase is a bioluminescent enzyme which is broadly used as a reporter protein in molecular biosensors. In this study, a novel luciferase with desired light emission wavelength and thermostability is reported. The results indicated that the new luciferase, namely super RLuc8, had a red-shifted spectrum and showed stable light emission. Super RLuc8 showed a 10-fold (p-value=0.0084) increase in the thermostability at 37°C after 20min incubation, in comparison to the native enzyme. The optimum temperature of the mutant increased from 30 to 37°C. Molecular dynamics simulation analysis indicated that the increased thermostability was most probably caused by a better structural compactness and more local rigidity in the regions out of the emitter site.

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