Part:BBa_K4144010
Improved recombined SAMe transporter opPet8p fused with linker
Usage and Biology
Linkers can be classified into three groups: flexible, rigid and cleavable. Flexible linkers are generally composed of small, non-polar or polar residues such as Gly, Ser and Thr. The most common is the (Gly4Ser)n linker (Gly–Gly–Gly–Gly–Ser)n, where n indicates the number of repeats of the motif. Polyglycine linkers have also been evaluated, but the addition of a polar residue such as serine can reduce linker–protein interactions and preserve protein function. Due to their flexibility, these linkers are unstructured and thus provided limited domain separation in a previous study.
We WHU-China plan to use such linker to contact opPet8p and sfGFP in order to avoid the opPet8p membrane localization disruption by adding a sfGFP tag.
Figure. 1 Linker addition design into opPet8p::sfGFP recombined protein
Application in our project
We performed fluorescent observation of the membrane localization of 6xHis::opMistic::opPet8p::linker::sfGFP, and set the old strain who expressed opPet8p without fusing with linker. We can obviously found that strains expressing opPet8p fused with linker have absolutely fewer particle-like inclusion bodies in cytosol compared with strains expressing opPet8p without linker (Fig 1). So we can conclude that the linker can work to facilitate the folding of proteins and reduce the formation of inclusion body. However, as the large copy number of vector and decrease of protein precipitation, large amount of proteins are distributed in cells whose fluorescent signal is too large to observe the membrane localization. Due to the lack of time, we haven’t explored more proper condition to observe, but we are optimistic to observe its membrane localization after decreasing the induction time.
Figure. 1 Comparison of fluorescent signals in strains expressing opPet8p fused with linker and without linker
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