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.
Team:NCTU Formosa/Improve
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 previous 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).
Improvement Part: (BBa_K2599016)
The improvement part that NCTU_Formosa 2018 modified contains the IPTG induced pT7 (BBa_I712074), strong ribosome binding site (BBa_B0034), Sf1a, GS linker (BBa_K1974030), snowdrop lectin (BBa_K1974020) and the 6X His-Tag (BBa_K1223006).
Introduction of μ-segestritoxin-Sf1a and Lectin
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 it is able to bind soluble ectracellular and intercellular glycoproteins.
Target Insects
Experiments and results
Preparation of Bio-insecticidal Proteins
We utilized Rosetta-gami DE3 strain to express both the previous part and improvement part. The proteins that was produced was then coated on leaves respectively and each leaf was placed inside containers with same number of larvae. The leaf remaining area was observed as shown as below.
Comparison of Plant Protecting Effect between Different Linker Design
Table 1: Comparison of plant protecting effects by changing the linker between protein Sf1a and lectin. (I): link proteins with GS linker (improvement part BBa_K2599016 ); (II): link proteins with AAA linker (previous part BBa_K1974022); (III): negative control group of Rosetta gami DE3 solution. After feeding for 7 hours, percentage of remained leaf area : improvement part > previous part > negative control.
Figure 1: Percentage of remained leaf area of improvement part, previous part and negative control.
In conclusion, through comparison, improvement group (I) is more effective in protecting leaf from larvae consuming than previous part (II) and inferred that elongation of linker can enhance the function of fusion proteins and provide a proper folding of proteins.
References
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.
