Difference between revisions of "Part:BBa K2817004"

Revision as of 13:00, 10 October 2019

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.

2019 NEU_CHINA

Characterization

Expressing vector has been constructed using pcold-1 backbone. We demonstrated SDS page and coomassie brilliant blue last year, but the result was not very convincing. So this time, western blotting is used to show the expression of myrosinase. Expressing vector harboring myrosinase gene is transformed into BL21 strain using chemical method. After incubating at 37℃ for 12h, CFU is inoculated to LB medium followed by 12h incubation. The culture is then diluted to OD600=0.2 and let grow to around 0.5. 1 mM IPTG is added to the culture to induce protein expression followed by 12h incubation. Cells are washed and collected, ready for western blotting. The molecular weight of protein myrosinase is 57.46 kDa.

Figure 1. Diagram for myrosinase expressing plasmid in pcold-1. Promoter cspA, a super strong promoter when incubating at low temperature. RBS, ribosome binding site, downstream is gene myrosinase.

Figure 2. Protein expression of myrosinase gene which transformed in BL21 strain. After induction of IPTG, the culture is incubated at 37℃ overnight. The concentration of protein loaded on this two lanes are different.

Sequence and Features