Difference between revisions of "Part:BBa K3139013"

Latest revision as of 00:12, 22 October 2019

Optimized anti-plasmodium fusion protein

Usage and Biology

The optimized anti-plasmodium fusion protein contains all the anti-plasmodium peptides uploaded (BBa_K3139000, BBa_K3139002,BBa_K3139003, BBa_K3139004,BBa_K3139005,BBa_K3139006, BBa_K3139007,BBa_K3139008, BBa_K3139009), TEV protease recognition and cleavage sites (BBa_K3139011) between each 2 peptide coding domains, and a His-tag is attached on the N-terminal for purification. They were found in some reviews in this field and chosen by our team members as their efficient effect reported. With reports of each sigle peptide, we primarily acquire the anti-plasmodium effect of each peptide. And we finally confirmed its sequence with help from mathmatical model. The copies and types of the peptides were selected elaborately.

Characterize

We transferred the plasmid into BL21 strain and cultured it in LB medium to obtain supernatant after ultrasonication. Then we purified our target His-tagged protein by using Ni-NTA magnetic beads. It was predicted that TEVp cleavage would not be so complete that nine different sizes of protein bands and TEVp's own bands would appear. In the preliminary experiment, we electrophoresised the purified protein on sodium dodecyl sulfate (SDS) -12.5% (wt/vol) polyacrylamide gel, followed by Western Blot to verify the expression and cleavage effect of this plasmid in the bacteria. In the result, the band of anti-plasmodium effector fusion protein (75,1kDa) in sample without TEVp is darker than the one in sample with TEVp, which strongly proves that our plasmid could express and be cleaved to produce single peptide.(Fig.1)

Fig.1 Preliminary verification of the effect of cleavage.
Lane 1: protein which purified from the sample of bacteria expressing effector and TEVp simultaneously.
Lane 2: protein which purified from the sample of bacteria expressing Effector only.

Figure.2 Mass spectrometry analysis of cleavaged fusion protein

The mass spectrometry analysis (Figure.2) shows that His-A, the smallest component of the fusion protein, was detected.

Sequence and Features