Part:BBa_K2155003
Contents
TalB---Carbon chain elongation
Usage and Biology
Transaldolase B is important for the balance of metabolites in the pentose-phosphate pathway. It can catalyze this reaction:
Sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate = D-erythrose 4-phosphate + D-fructose 6-phosphate
In our project, we use this enzyme to condense Glycolaldehyde and Dihydroxyacetone to D-xylulose(Figure1 and Figure2).If you use it to catalyze reaction in vivo, It needs one cofactor, thiamin diphosphate.
Figure 1. D-xylulose was synthesized from glycolaldehyde and DHA by TalB.
Figure 2. HPLC analysis of samples from in vitro experiment of TalB.
Figure 3. TalB is fused with dockerin and then it can bind with matching CcsCohesin.
TalB was cloned into the plasmid vector pET22b+ for expression. We purify this protein by His tag. This year we fused TalB with CcsDock(Figure3), and then CcsDock could bind with CcsCohesin(Figure 4), which is part of our scaffold protein. Therefore, our core enzyme could form a enzyme complex, and the catalytic efficiency could be greatly improved.
Cultivation, Purification and SDS-PAGE
Cultivation
The part was assembled with T7 promoter and RBS in pET28a plasmid vector for expression. E. coli strain BL21(DE3) harboring the appropriate plasmid was grown at 37 °C in 2xYT medium overnight with suitable concentration of antibiotic. When the culture was grown at 37°C to an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were grown for 16h at 16°C.
Purification
Cells were centrifuged at 5500rpm for 15min at 4°C. Resuspend the cell paste expressing recombinant protein in binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1mM β-mercaptoethanol, pH7.4). Disrupt the cells with sonication for 20 min with suitable power on ice and centrifuge at 8000 rpm for 40 min at 4°C. Remove remaining particles by passing the supernatant through a 0.45μm filter. The HisTrap™ column (GE Healthcare, Inc.) was equilibrated with binding buffer. Load the sample and wash the column with binding buffer. Elute the target protein with a concentration gradient of imidazole starting with binding buffer including 50mM imidazole and ending with the same buffer including 300mM imidazole. The eluted fraction containing the target protein were concentrated by Column-PROTEIN-Concentrate™ with a 10 kDa cutoff.
SDS-PAGE
Protein purification was checked by SDS-PAGE and the resulting protein is quantified by Coomassie (Bradford) Protein Assay. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 861
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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