Difference between revisions of "Part:BBa K2910000:Design"
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===Design Notes=== | ===Design Notes=== | ||
Due to the use of a vector with the N. terminal pelB leader sequence (pET22(B)) and unknown contributions of the N terminal signal sequence to PETase activity, we wanted to avoid N-terminal hexahistidine tagging of IsPETase. We selected C terminal tagging to enable protein purification with a nickel-based resin (which binds the hexahistidine tag). The two mutations, W159H and S238F, were based on the PNAS publication by Austin et al, 2018 in which mutation of these residues was reported to enhance the catalytic activity of PETase. | Due to the use of a vector with the N. terminal pelB leader sequence (pET22(B)) and unknown contributions of the N terminal signal sequence to PETase activity, we wanted to avoid N-terminal hexahistidine tagging of IsPETase. We selected C terminal tagging to enable protein purification with a nickel-based resin (which binds the hexahistidine tag). The two mutations, W159H and S238F, were based on the PNAS publication by Austin et al, 2018 in which mutation of these residues was reported to enhance the catalytic activity of PETase. | ||
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Latest revision as of 02:25, 22 October 2019
IsPETase-W159H-S238F with C-terminal Hexahistidine Tag
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 67
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Due to the use of a vector with the N. terminal pelB leader sequence (pET22(B)) and unknown contributions of the N terminal signal sequence to PETase activity, we wanted to avoid N-terminal hexahistidine tagging of IsPETase. We selected C terminal tagging to enable protein purification with a nickel-based resin (which binds the hexahistidine tag). The two mutations, W159H and S238F, were based on the PNAS publication by Austin et al, 2018 in which mutation of these residues was reported to enhance the catalytic activity of PETase.
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Source
Protein sequence was retrieved from Genbank (NCBI) under GAP38373.1, corresponding to the sequencing of Ideonella sakaiensis strain 201-F6 by Yoshida et al (2016). This amino acid sequence was subsequently converted to DNA sequence using the EMBOSS-Backtranseq algorithm (https://www.ebi.ac.uk/Tools/st/emboss_backtranseq/) and codon-optimized for E. coli K-12. Mutations were created manually in Benchling.