Difference between revisions of "Part:BBa K1582015"

Latest revision as of 02:52, 25 September 2015

BFP+sJanus-m Fusion Protein

Sequence and Features

Usage

Admittedly the separation of proteins is difficult in biological experiments. More often than not, we use chromatography, but it is expensive. Extraction turns to be cheap, however, its low separation rate is really an obstacle. </br> Here comes the Janus, a kind of amphipathic protein, which is made into a fusion protein with the target and carry it to leave the bulk protein phase. We could use aqueous two-phase systems to make them separated from mixture, according to the property that Janus will direct to the phase of detergent in the system of detergent/polymer. BFP-inJanus fusion protein could be used to verify this purification system. </br>

Biology

mTagBFP is a monomeric protein with a narrow fluorescence emission spectrum with a maximum at 456 nm. It has a tyrosine-based chromophore, giving it substantially higher brightness, faster chromophore maturation and higher pH stability than blue fluorescent proteins with a histidine in the chromophore. </br> sJanus are small secreted fungal proteins which can be found in filamentous fungi, which play a role in a broad range of processes in the growth and development of filamentous fungi. They shows thread-like structure, can be expressed in prokaryotic cells like E.coli, and are involved in transferring themselves to detergent phase spontaneously. </br> We did some mutations to it, and we call it sJanus-m.</br>

Reference

[1]Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands. Hydrophobins: multipurpose proteins. Annu Rev Microbiol. 2001;55:625-46.

Protein Expression

We insert the plasmid into competent C+ to make the fusion protein secreted.</br> Express the fusion protein:</br>

Purify the fusion protein:

Figure 19. BFP- sJanus-m: The target protein is about 40kDa. As is shown in the picture, the protein is purified through Ni-chelating affinity chromatography.