Difference between revisions of "Part:BBa K3771006"

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JJU-6xHis

Description

JJU (cysteine lyase) is an enzyme weighing approximately 43.1 kDa. JJU catalyzes the beta replacement reaction of L-cysteine and sulfite to form L-cysteate and hydrogen sulfide[1].

Biology

The three taurine production pathways incorporated into our E. coli include the L-cysteine sulfinic acid pathway, L-cysteine sulfonic acid pathway, and the JJU-CoaBC pathway[1,2].

JJU10 is an enzyme in the JJU10-CoaBC pathway, one of three possible taurine synthesis pathways. Its main function is to convert L-cysteine to L-cystate.

Usage

JJU10 enzyme was used in vitro testing of taurine production. The sequence for JJU10 enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC). A 6xHis-tag is added to the C-terminal of the JJU10 protein, allowing for confirmation of JJU10 expression by western blot using the anti-6xHis antibody.

Characterization

The JJU10-6xHis fragment was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.

Fig. 2 Confirmation of jju fragment by PCR. M: Marker; Lane 1: jju (1180bp)

Fig. 3 SDS-PAGE of the JJU10 enzyme to confirm protein expression.

Taurine production yield of JJU10 with other production enzymes calculated by high-performance liquid chromatography (HPLC).

Taurine production of JJU in BL21(DE3) and CoaBC in BL21(DE3) in supernatant and whole cell samples.

References

BRENDA - Information on EC 4.4.1.10 - cysteine lyase. Brenda-enzymes.org. Published 2021. Accessed October 6, 2021. https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10

Sequence and Features