Revision as of 14:14, 21 October 2019

Coding sequence for W159H/S245I IsPETase double mutant

A coding sequence for mutated W159H/S245I PETase from Ideonella sakainesis. It is codon optimized for Escherichia coli.

Usage and Biology

A coding sequence of the PETase double mutant W159H/S245I.

This sequence is modified from a sequence for wild type PETase which was also codon optimised for Escherichia coli, obtained from previous studies done on PETase.[1]

Origin and biology

The enzyme is a hydrolase which degrades polyethylene terephthalate into simple molecules: MHET, BHET, and TPA by cleavage of the ester bond within the polymer. It was originally found in the bacteria Ideonella sakaiensis, which uses PET as a carbon source, and integrates the degradation products into its metabolic cycle.

Design

We analyze the rationale for PETase mutant design from previous studies done on the residue modification of this enzyme. A clear trend in most successful mutation attempts is that an increase in hydrophobicity or a binding site similar to T. fusca cutinase, which is narrower.

The mutation sites locate in substrate binding site, subsite II where three MHET moieties are bound through hydrophobic interaction.

In TfCut2, Histidine 169 residues and Isoleucine 253 are located at the corresponding positions of Trpytophan 159 and Serine 245 in subsite II of IsPETase. The resulting double mutant makes the substrate binding site, substrate II more cutinase-like and increases the hydrophobic property of the enzyme.

Characterisation

In our experiments, to insert this gene into cells, the PET-21b vector is used due to its high copy number and the presence of T7 promoter and a lac operon. We use DH5ɑ as host cells due to its high insert stability. Then, extracted DNA is transformed into C41(DE3) cells, which we use to perform the protein induction due to the toxic nature of PETase.

After the protein is induced using 0.05mM IPTG, it can be purified and extracted using a column with nickel resin due to a 6X His-Tag fused with PETase outside the globular structure. After purification, SDS-PAGE can be performed to confirm successful expression. Figure 1: SDS-PAGE of purified W159H/S245I PETase. A band of around 30 kDa is clearly shown

As shown above, the thick band around 30 kDa shows successful expression of the construct.

After protein purification, an enzyme assay is performed to confirm the protein activity.

Sequence and Features