Experiments

Construct Design

In order to analyze the function of PETase mutants in-vitro, our team decided to use a pET22b plasmid vector. Since proper folding of PETase occurs in the E. coli periplasm, pET22b was chosen because it contains a pelB leader sequence (Singh et al. 2013), which allows for the periplasmic targeting of PETase, followed by signal cleavage via the periplasmic protein, signal peptidase (Singh et al. 2013). Furthermore, this vector allows for IPTG-induction of PETase production via the presence of the LacI promoter. PETase variants were synthesized and cloned into PET22b courtesy of GenScript.

LOBSTR Cells

When conducting His-tagged purification of recombinant proteins from E.coli, naturally histidine rich proteins may be pulled down with the protein of interest. The most common of these proteins are arnA and slyD. Low Background Strain (LOBSTR) is a strain of E.coli similar to BL21. It grows at a similar rate to standard BL21, but contains mutants of slyD and arnA with lower affinity to Nickel-NTA resins. This allows for greater sample purity without the need of multiple purification steps, by eliminating arnA and slyD presence when eluting. (Andersen et al. 2013).

Bacterial Chases

All of our plasmids were transformed into and purified from DH5alpha competent cells. For protein purification, transformation into LOBSTR cells was necessary to prevent the presence of unwanted proteins that have affinity to Nickel-NTA resins.

Protein Purification

We used immobilized metal-ion affinity chromatography to isolate our HIS-tagged protein of interest from LOBSTR cells. The Hexa-His-tag is a chain of six histidines that are attached to the C-terminus end of the our PETase enzyme. Histidine has an imidazole side chain that has high affinity for Ni^2+, hence Nickel-NTA resin is bound to the affinity chromatography column. The LOBSTR with all of the proteins is lysed by sonication and lysozyme, and spun-down to isolate all cellular proteins. Proteins are introduced into the column; the proteins of interest with the His-tag will bind to the Nickel-NTA resin. Washes with a slightly higher concentration of imidazole are completed to eliminate proteins not bound to Nickel-NTA. Finally, elutions are performed whereby a solution with excess imidazole is added to the column. This will complete with the His-tag to binding with the resin, dislodging the protein of interest and a relatively pure protein should be obtained in the end (Bornhorst, 2000).

Quantification

Following protein purification and SDS-PAGE, protein concentration was quantified by performing Bradford Assays in serial dilutions.

PETase Activity Assays

To test the catalytic activity of our variants, a p-nitrophenyl butyrate assay was performed. This assay is designed to test for activity of certain hydrolases. The molecule consists of p-nitrophenol bonded to butyric acid in an ester linkage. Hydrolysis of the ester releases p-nitrophenolate (the alcohol is deprotonated), which is a chromophore than can be measured spectrophotometrically at 405 nm (Pliego et al., 2015). The p-nitrophenyl butyrate assay, which has been used to measure PETase activity in previous studies (Furukawa et al., 2018) was chosen because its structure is similar enough to PET subunits to be recognized by PETase and cleaved; thus, p-nitrophenolate production can be used as an indicator of enzymatic activity.

References

1.) Pliego, J., Mateos, J. C., Rodriguez, J., Valero, F., Baeza, M., Femat, R., Camacho, R., Sandoval, G., Herrera-López, E. J. (2015). Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate. Sensors (Basel, Switzerland), 15(2), 2798–2811. doi:10.3390/s150202798
2.) Furukawa, M., Kawakami, N., Oda, K., Miyamoto, K. (2018). Acceleration of Enzymatic Degradation of Poly(ethylene terephthalate) by Surface Coating with Anionic Surfactants. Chemsuschem, 11(23), 4018-4025. doi:10.1002/cssc.201802096
3) Andersen KR, Leksa NC, Schwartz TU. (2013). Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins. 81(11):1857-61.
4) Seo, Hogyun, et al. “Production of Extracellular PETase from Ideonella Sakaiensis Using Sec-Dependent Signal Peptides in E. Coli.” Biochemical and Biophysical Research Communications, vol. 508, no. 1, 2019, pp. 250–255., doi:10.1016/j.bbrc.2018.11.087.
5) Singh, Pranveer, Likhesh Sharma, S. Rajendra Kulothungan, Bharat V. Adkar, Ravindra Singh Prajapati, P. Shaik Syed Ali, Beena Krishnan, and Raghavan Varadarajan. “Effect of Signal Peptide on Stability and Folding of Escherichia Coli Thioredoxin.” PLoS ONE8, no. 5 (July 2013). https://doi.org/10.1371/journal.pone.0063442.
6) Bornhorst, Joshua A., and Joseph J. Falke. “[16] Purification of Proteins Using Polyhistidine Affinity Tags.” Methods in Enzymology Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification, 2000, 245–54. https://doi.org/10.1016/s0076-6879(00)26058-8.

Protocols

The following were the protocols used this year:
Gibson Assembly
Midi Plasmid Purification
Mini Plasmid Purification
Transformation
SDS Page
Plate Preparation
p-Nitrophenol Butyrate assay
Protein Purification
Competent Cells
Gel Extraction
Bradford Assay
Restriction Enzyme Digest

Parts

You can find the parts used in our project [here]

Team:Toronto/Experiments