Part:BBa_K2239001

7alpha-HSDH-CBD (T7 promoter-lac operator-RBS-Histag-7alpha HSDH-CBD-T7 terminator) Construct This device codes for the 7alpha-HSDH-CBD fusion protein.

The vector of 7alpha-HSDH for its expression is PET-28x, and is formed by modifying PET-28a’s restriction enzyme sites EcoR I and Xba I. The 7alpha-HSDH sequence is cloned from the genome of E.coli through PCR amplification, using the primers designed and synthesized based on its sequence. The restriction site BamH I is added to the upstream primer, and Hind III is added on the downstream primer. The CBD sequence is retrieved from the GeneBank, and is inserted into plasmid pUC57. The CBD gene is then cloned from the plasmid by PCR, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.

We first insert 7alpha-HSDH into the modified PET-28x at BamH I and Hind III, and CBD at Hind III and Xhol I. Then the whole gene fragment, T7 promoter-lac operator-RBS-Histag-7alpha HSDH-CBD-T7 terminator, is retrieved from this plasmid by using PCR, with EcoR I and Xba I added on its upstream primer, and Pst I and Spe I on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.

[Fig. 1. pSB1C3-CBD-7alpha-HSDH]

Proof of function through experiments 7alpha-HSDH catalyzes the oxidation of steroids at C-7 position. It catalyzes the oxidation of CDCA into intermediate product 7-oxo-LAC (7-ketolithocholic acid), where the hydroxyl at C-7 position becomes carbonyl. CBD protein binds to cellulose. Connected with 7alpha-HSDH, CBD immobilizes enzyme 7alpha -HSDH after expression, by binding to the cellulose on gauze.

7alpha-HSDH Enzyme activity test: The activity of 7alpha-HSDH is characterized by using spectrophotometer at 340nm. 7alpha-HSDH is reacted with CDCA in the presence of NAD+. The activity of 7alpha-HSDH is calculated by measuring the change of absorbance value at 340nm caused by the NADH produced. The reaction solution is mixed with a piece of gauze when measuring enzyme activity after immobilization. The reaction system of three different enzyme concentrations includes: H2O: 2.7ml 1.5mol tris (ph8.0): 0.3mL 22mg/mL NAD+: 40μL CDCA: 10μL; 7alpha -HSDH: 30/60/90μL; H2O:120μL

Results: Enzyme activity before immobilization: [Fig. 2. Activity of 7alpha-HSDH before immobilization] Enzyme activity after immobilization: [Fig. 3. Activity of 7alpha-HSDH after immobilization]