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CBD-7alphaHSDH

CBD--7alpha-HSDH (T7 promoter--lac operator--RBS--His-tag--7alpha-HSDH--CBD--T7 terminator)

This device codes for the 7alpha-HSDH--CBD fusion protein.

Construct

The vector of 7alpha-HSDH--CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.

The 7alpha-HSDH sequence is cloned from the genome of E.coli DH5alpha 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 to the downstream primer.

The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.

Firstly, the 7alpha-HSDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--7alpha-HSDH--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.

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

Usage and Biology

7alpha-HSDH(7alpha-hydroxysteroid dehydrogenase) catalyzes the oxidation of hydroxysteroids at C-7 position and back, as it converts NAD+ to NADH and back. It catalyzes the oxidation of CDCA into intermediate product 7-oxo-LAC (7-ketolithocholic acid), where the hydroxyl at C-7 position becomes carbonyl.[1]

CBD (cellulose binding domain) is able to bind to cellulose. When connected to 7alpha-HSDH, CBD is able to immobilize the enzyme 7alpha-HSDH after expression, by binding to the gauze inside the solution on its cellulose.[2]

The function of cellulose binding domain

The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is left after washing the gauze mixed with GFP-ChBD (Chintin binding domain).

[Fig. 2. GFP-CBD on gauze before washing]

[Fig. 3. GFP-CBD on gauze after washing]

[Fig. 4. GFP-ChBD on gauze before washing]

[Fig. 5. GFP-ChBD on gauze after washing]

Expression and Immobilization[2]

The constructed pET28x--7alpha-HSDH--CBD plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.

The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece and the gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the cellulose on gauze, and the enzyme is successfully immobilized.

Enzyme Activity

The oxidation of CDCA to 7-OXO-LCA using 7alpha-HSDH

[Fig. 6. Reaction process]

7a-HSDH from Ecoli 5a is an NAD+ dependent enzyme. The 3 mL reaction consists of 150mM phosphate buffer(pH 8.0), 10mM CDCA, 0.2mM NAD+. The reaction started when the solution is combined with 7a-HSDH-CBD-enzyme-binding gauze that in different concentration, includes 60ul, 120ul, and 180ul liquid supernatant of ultrasonication bacteria solution. The control group was testify under the same solution and condition but using pET28x-CBD liquid supernatant of ultrasonication bacteria solution to bind with gauze in the concentration of 180ul. Before adding the gauze into the solution, the gauze was washed by ddH2O for 3 times in order to purify the enzyme.

The CDCA was convert to 7OXO-LCA by loosing a pair of hydrogen(2H+and 2e-) from the 7-hydroxyl group and form 7-carbonyl group.The co-enzyme NAD+ is the acceptor of the a pair of hydrogen(2H+and 2e-) and e-, and was transformed into NADH. The enzyme activity was determined spectrophotometrically atv340 nm (ε = 6.22 mM-1 cm-1) and room temperature. One unit of activity is defined as the amount of enzyme catalyzing the synthesize of 1 mmol of NADH per min under the assay conditions used.

Result

[Fig. 7. Result]

Oxidation of CDCA to 7-oxo-LCA using E. coli 7alpha-HSDH and NAD+ regeneration

[Fig. 8. Reaction process]

CDCA was converted in a 3mL solution containing 150 mM phosphate buffer(pH 8.0), 10 mM CDCA, 30 mM sodium pyruvate, 0.25mM NAD+, combined with 3U/ml LDH and1 1 U/ml E. coli DH5a 7a-HSDH-CBD at room temperature.

The bioconversion experiments were monitored via HPLC measurements. The sample was analyzed by UV detection at 210 nm, using a mobile phase of methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using C18.

Result

As shown in figure, the transformation was complete after 2 h. The final yielding rate is 94% with REM 2.7%.

Reference

[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.

[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov: Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.

Sequence and Features