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DESCRIPTION

This year, our goal is to engineer a thermostable protein using the SpyRing technique1. This involves cloning the protein of interest between the SpyTag at the N-terminus and the SpyCatcher at the C-terminus. The IPTG-regulated, T7-promoter-based vector (such as pET28a) serves as an optimal vector for gene expression in E. coli BL21. First, in this context, we explored the BioBrick gene bank for existing parts of GFP gene (i.e., T7-GFP) and assessed their thermostability. Next, we engineered a SpyTag-GFP-SpyCatcher construct (i.e., T7-SpyTag-GFP-SpyCatcher) and evaluated its thermal resistance. Lastly, we introduced a point mutation in the SpyTag domain (i.e., T7-SpyTag D7A-GFP-SpyCatcher), which impedes circular protein formation, and then studied the resultant protein structure and its effect on thermal resistance.




With this technique, we engineered several potential lipase genes that could degrade PCL plastics. Two of these (PCLase I and PCLase II)2 are novel discoveries from Dr. Fan Li, while the other two (BCLA3 and CALB4) are commonly cited in numerous studies and are commercially available. After using the pNPB assay, a standard test for lipase activity, PCLase I showed the highest activity in our lab. We then characterized SpyTag and SpyCatcher-incorporated PCLase I and its corresponding mutant in detail, including the thermostability test using the pNPB assay, protein structure analysis by SDS-PAGE and Coomassie Blue staining, and the PCL degradation properties.



COLLECTION TABLE

References

  1. Reddington SC, Howarth M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol. 2015 Dec;29:94-9. doi: 10.1016/j.cbpa.2015.10.002. Epub 2015 Oct 30. PMID: 26517567.
  2. Li L, Lin X, Bao J, Xia H, Li F. Two Extracellular Poly(ε-caprolactone)-Degrading Enzymes From Pseudomonas hydrolytica sp. DSWY01T: Purification, Characterization, and Gene Analysis. Front Bioeng Biotechnol. 2022 Mar 18;10:835847. doi: 10.3389/fbioe.2022.835847. PMID: 35372294; PMCID: PMC8971842.
  3. Yang J, Guo D, Yan Y. Cloning, expression and characterization of a novel thermal stable and short-chain alcohol tolerant lipase from Burkholderia cepacia strain G63. Journal of Molecular Catalysis B: Enzymatic. 2007 Jan 9;45(3-4):91-96. doi:10.1016/j.molcatb.2006.12.007
  4. Ujiie A, Nakano H, Iwasaki Y. Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli. J Biosci Bioeng. 2016 Mar;121(3):303-9. doi: 10.1016/j.jbiosc.2015.07.001. Epub 2015 Aug 10. PMID: 26272415.