Part:BBa_K2599016
T7 Promoter+RBS+Sf1a+GS linker+snowdrop-lectin+linker+6X His-Tag
Introduction
A fusion protein is composed by joining of at least two domains that are encoded by separate genes and finally translated as a single polypeptide. And the linker that connects protein domains often plays an important role.
To enhance the function of fusion proteins and provide a proper folding of proteins, NCTU_Formosa 2018 modified the linker between Sf1a (spider toxin) and lectin (orally active protein) by elongating short linker AAA (3 a.a.) to GS linker (18 a.a.).
Modifying and Improving the Existed Biobrick
Previous Part:(BBa_K1974022)
The existing part from NCTU_Formosa 2016 contains the IPTG induced PT7 (BBa_I712074), strong ribosome binding site (BBa_B0034), Sf1a, AAA linker, snowdrop lectin (BBa_K1974020) and the 6X His-Tag (BBa_K1223006).
Figure 1. Previous part
Improvement part
In many application, the fusion proteins consisting of multiple protein domains is a popular and highly successful approach to engineering new protein functions. The linker that connects the fusion protein often plays an important role in fusion proteins. Therefore, we decided to change the linker between Sf1a and lectin to try whether the function of this protein become stronger. The GS linker we used in this improvement part was also provided by NCTU_Formosa 2016. It contained 18 amino acid sequence, which is a Gly-Gly-Ser repeated linker. We utilized this linker to optimize the fuction of Sf1a and lectin.
Figure 2. Improvement part
Introduction
μ-segestritoxin-Sf1a is kind of insecticidal toxin, contains three disulfide bonds. It will inhibits insect voltage-gated sodium channels by blocking the channel pore.
Lectin is carbohydrate-binding proteins, and is able to bind soluble ectracellular and intercellular glycoproteins.
Target Insect
Result
1. Comparison of Plant Protecting Effect of Different Design of Linker between Protein Sf1a and Lectin
Figure 4. Comparison of plant protecting effects
(I): Fusion proteins with GS linker (Improvement part BBa_K2599016 ); (II): Fusion proteins with AAA linker (Previous part BBa_K1974022); (III): Negative control group of Rosetta gami DE3 solution. After feeding for 7 hours, leaf area of (I) remained unchanged compared with leaf remaining area after 3 hours; While leaf of (II) and (III) was continued consumed by larva. Through comparison, improvement group (I) is more effective in protecting leaf from larvae consuming than previous part (II).
2. Plant Protecting Effect of sequence Sf1a-GS Linker-Lectin (Improvement Part) in pET-32a
Figure 4. Plant protecting effects by inserting the improvement part into pET-32a.
Through comparison, improvement part with the highest concentration shown the most effective as it had the largest leaf area remaining.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
1. Elaine Fitches, Martin G. Edwards, Christopher Mee, Eugene Grishin, Angharad M. R. Gatehouse, John P. Edwards, John A. Gatehouse “Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion,” Journal of Insect Physiology, 2004, 50, pp.61-71
2. Elaine C. Fitches, Prashant Pyati, Glenn F. King, John A. Gatehouse, “ Fusion to Snowdrop Lectin Magnifies the Oral Activity of Insecticidal Omega-Hexatoxin-Hv1a Peptide by Enabling Its Delivery to the Central Nervous System,”
3. Monique J. Windley, Volker Herzig, Slawomir A. Dziemborowicz, Margaret C. Hardy, Glenn F. King and Graham M. Nicholson, “Spider-Venom Peptide as Bioinsecticide,” Toxins Review, 2012, 4, pp. 191-227.
4. A. Lipkin, S. Kozlov, E. Nosyreva, A. Blake, J.D. Windass, E. Grishin (2001, April 9). Novel insecticidal toxins from the venom of the spider Segestria florentina. Toxicon, 40, 125-130.
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