Difference between revisions of "Part:BBa K4488013"

Revision as of 08:06, 11 October 2022

Fusion of free-use GFP with CBDcipA (cellulose-binding domain) at the C-terminal end with a linker

The construct can be cloned into an expression vector such as pET28c in E.coli to produce a fusion protein of fuGFP with CBDcipA. The fuGFP sequence is towards the N terminus of the protein with CBDcipA (BBa_K4488024 ) downstream followed by a stop codon. Recognition sites for BamHI and BsaI are present before the RBS allowing golden gate cloning. XhoI and BsaI are also present downstream of the stop codon.

Usage and Biology

Our project provided preliminary evidence that CBD and cellulose can be used to purify proteins by using fuGFP-linker-CBDcipA.

Figure 1: Fluorescence measurements of filter paper binding test with fuGFP-linker-CBDs. 200 uL of TOP10-pUS250v3-fuGFP-CBD cell lysate was added to a 5 mm diameter paper filter disk and incubated for 1 h before washing with 200 uL NT buffer. Fluorescence measurements show that lysate with fusion proteins with the added linker exhibit more fluorescence than control cell lysate and makes the filter disks fluorescent.. fuGFP-linker-CBDcipA causes the greatest gain in fluorescence by paper filter disks.

The linker improved the solubility and fluorescence of the sequence. Below is the fluorescence assay depicting a higher reading compared to our previous construct BBa_K4488009 :

Figure 2: Fluorescence readings of fuGFP-linker-CBDs compared to fuGFP-CBDs in different E.coli plasmids. The uninduced and induced plasmids are also compared.

Furthermore, expression using the BL21(DE3)-pET28c(+) system results in high yields of protein which can be purified by binding to cellulose and eluting using glucose.

Figure 3: SDS-PAGE of fuGFP-linker-CBDcipA eluted from microcrystalline cellulose with glucose (1 M) (top). Mass spectrometry results confirm the identity of the band outlined in red to be fuGFP-linker-CBDcipA (bottom). The single distinct band outlined in red is observed at around 46 kDA in the elution fraction corresponding to our protein of interest. Furthermore, the amount of fusion protein bound to cellulose is almost halved after 4 elutions with glucose.

Sequence and Features