Part:BBa_K2817004
Myrosinase (horseradish)
Myrosinase is present in cruciferous plants such as broccoli, Brussels sprouts and Celery cabbage. It can convert the precursor glucosinolates in cruciferous plants into sulforaphane, a well-known anticancer substance. Sulforaphane is an activator of Nrf2, which induces Nrf2 activation and translocation into the nucleus, binding to the antioxidant response element (ARE) to promote transcriptional activation of the phase II metabolic enzyme gene. As a result, sulforaphane, on the one hand, can eliminate cancer cells by inducing phase II reaction; on the other hand, it can alleviate inflammation by inducing antioxidant enzymes (such as HO-1 and SOD) to resist oxidative stress. The horseradish enzyme derived from horseradish has good catalytic activity at body temperature and in vivo pH.
Usage and Biology
We inserted this part into plasmid pet-28a. Next, we transformed the plasmid of myrosinase-His into BL21, and cultured at 37 ℃ overnight and diluted to OD = 0.2. After growth for 2 h at 37 ℃, different concentrations of IPTG were added to induce at 16 ℃ for 16 h. Subsequently, bacterial cell lysate was obtained and the expression of myrosinase was detected by SDS-PAGE. (Figure 1). The sequence of the myrosinase enzyme was optimized based on the K12 strain and the position of the strip is correct, so myrosinase was successfully expressed. The myrosinase can convert cruciferous vegetables contained glucosinolates into sulforaphane which has well-known anti-cancer activity. A previous work demonstrated the activity of myrosinase expressed by E. coli Nissle 1917 in vivo and in vitro, the expressed myrosinase in our constructed plasmid should with broad applications in vivo.
Figure 1. SDS-PAGE analyses on bacterial lysate to detect myrosinase. Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.25mM IPTG; Lane3: 0.5mM IPTG, Lane4: 0.75mM IPTG induction for 16h at 16℃.
[1] Ho CL, Tian HQ.et al. 2018 Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nature Biomedical Engineering. 2, pages27–37
[2] Tortorella, S. M., Royce, S. G., Licciardi, P. V. & Karagiannis, T. C. Dietary sulforaphane in cancer chemoprevention: the role of epigenetic regulation and HDAC inhibition. Antioxid. Redox Signal. 22, 1382–1424 (2015).
[3] Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang MI, Kobayashi A, Yamamoto M, Kensler TW; Talalay P. Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keapl sensor modified by inducers. Proc Natl Acad Sci U S A. 2004;101(7):2040一2045.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 139
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 368
Illegal BsaI site found at 1397
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