Part:BBa_K2938004
P20
Description
p20 is a 20 kDa protein which can be isolated from Bacillus thuringiensis subsp. israelensis (Bti). It is a chaperone, which increases the production of Cry11Aa while expressed, and leads to higher mosquito larvicidal activity [2].
Amplification
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 264
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 264
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 264
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 264
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 264
- 1000COMPATIBLE WITH RFC[1000]
Contribution by iGEM Guelph 2022: The effect of P20 on cell viability and stability of Cyt1Aa
Bacillus thuringiensis subsp. Israelensis encodes crystal (Cry) and cytotoxic (Cyt) proteins which lyse target insect cells through a similar process. The expression of these Cry and Cyt proteins can be dependent on the coexpression of accessory helper proteins (Shao et al., 2001). In this case, the helper protein P20 increases the expression of Cry proteins (Cry1Ac, Cry4Aa, Cry4Ba, and Cry11Aa) and is necessary to observe colony formation in its original host and E.coli (Shao et al., 2001).
The P20 helper protein is a 20 kDa protein that was first identified in 1987 by Mclean and Whiteley and following this discovery, it was hypothesized that this protein allows for the crystalization of Cyt1Aa, Cry4Ba, Cry11Aa, and Cry4Aa. Manasherob et al. (2001), later reported that plasmid constructs expressing only Cyt1Aa showed reduced colony-forming ability and Cyt1Aa protein expression (Manasherob et al., 2001). While Cyt1Aa and P20 expressing plasmid constructs showed higher Cyt1Aa protein expression and cell viability in recombinant E.coli cells (Manasherob et al., 2001). This confirms that P20 stabilizes Cyt1Aa and inhibits cell death. Although the exact mechanism by which P20 does this was not known in 1987, Manasherob et al. (2001) proposed that pre-mature activation of Cyt1Aa and lethality was a result of incomplete degradation of this Cyt protein. P20 binds to and prevents the degradation of Cyt1Aa, which ultimately results in the viability of the cell (E.coli and Bacillus thuringiensis cells) (Manasherob et al., 2001). This is an important step in the expression of Cyt1Aa because this Cyt protein does not form inclusion bodies that further protect the cell from Cyt1Aa’s cytotoxic effects (Manasherob et al., 2001). For these reasons, the coexpression of P20 must be considered when expression of Cyt1Aa is desired as this protein stabilizes the crystal formation and protects cells from the lethal effects of this protein.
References
Manasherob, Robert, et al. “Effect of Accessory Proteins P19 and P20 on Cytolytic Activity of cyt1aa from Bacillus Thuringiensis Subsp. Israelensis in Escherichia Coli.” Current Microbiology, vol. 43, no. 5, 3 Apr. 2001, pp. 355–364., https://doi.org/10.1007/s002840010316.
Shao, Z., Liu, Z., & Yu, Z. (2001). Effects of the 20-kilodalton helper protein on cry1ac production and spore formation in bacillus thuringiensis. Applied and Environmental Microbiology, 67(12), 5362–5369. https://doi.org/10.1128/aem.67.12.5362-5369.2001.
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