Part:BBa_K2915150:Experience

AFP-lu1 production

We were able to insert the TAL2 gene into a plasmid containing the T7 promoter and RBS which is a psB1C3 vector. SDS-PAGE was performed and stained with Coomassie blue using cells containing this Biobrick, to verify the production of our protein we did different growing conditions to determine the best growing conditions by using two types E.coli bacteria : -DH5a -BL21 DE3

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Figure 1. SDS-PAGE of the production in DH5a and BL21 DE3 of AF-lu1, well 1 and 2 are induced at 30°C with 0.1M IPTG for 3hours in DH5a and BL21 DE3, respectively. Well 5 and 6 are not induced of AF-lu1 in DH5a and BL21 DE3 respectively. Well 3 and 4 are induced at 16°C with 0.1M IPTG overnight in DH5a and BL21 DE3, respectively.

AFP-lu1purification

After having realized the AFP-lu1 production tests, the purification was realized in order to obtain pure protein fractions. For this, the E. coli BL21 cells were lysates and then are purified. Purification of the protein was realized with a HisTag column which is a manual chromatography system for quick, easy ad small quantity purification. The fusion of the histine tag with AFP-lu1 allows to separate our protein from the other proteins.

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Fig 2. SDS-PAGE of AFP-lu1 after purification with HisTag column. LD= loading sample, NR= non-restraint, E1 to E7 correspond to the eluted fractions.

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Fig 3. Western Blot analysis of AFP-lu1 after purification with HisTag column. (a) LD= loading sample, NR= non-restraint, E1 to E3 correspond to the eluted fractions. (b) from E4 to E7 correspond to the eluted fractions.

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