Part:BBa_K3075000

PAM-SnoopT

Introduction

PAM-SnoopT consists of the enzyme Phenylalanine aminomutase (PAM) fused at the C-terminus to a short polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of PAM originating from Taxus wallichiana var. chinensis was utilised. (1)

Usage and Biology

Phenylalanine aminomutase catalyses the conversion of 2S-α-phenylalanine to 3R-β-phenylalanine. (2) Naturally, this enzyme is involved in the synthesis of trans-cinnamate from L-phenylalanine, an important part of Phenylpropanoid metabolism. It is also known to catalyse the initial step of N-benzoyl phenylisoserinoyl biosynthesis, which functions as the side chain of the anticancer drug Paclitaxel. (1) Recombinant phenylalanine aminomutase has a sequence of 698 amino acid residues with a molecular mass of 76,530 Daltons and maintains stability at a pH of 9-11 and temperature of 60-70C.

Characterisation

The PAM-SnoopT gBlock was synthesised by IDT. PAM-SnoopT was ligated into pET19b plasmid backbone by Gibson assembly and transformed into competent T7 express E.coli cells. Colony PCR was performed using primers listed below and the amplicon was visualised by gel electrophoresis.

Primers used:

- T7 Forward : 5’-TAATACGACTCACTATAGGG

- T7 Reverse : 5’-GCTAGTTATTGCTCAGCGG

A small scale grow up of colonies was performed and plasmid DNA was extracted via a QIAGEN miniprep kit. Miniprepped samples were visualised via gel electrophoresis (Figure 1) and submitted for sequencing by the Ramaciotti Centre for Genomics (Figure 2).

Starter culture was made for colonies of interest and large-scale grow up was done. Expression of the recombinant protein was induced using IPTG and harvested cells were lysed via Sonication. The His-tagged protein was then purified via IMAC. Results are shown on figure 3.