Part:BBa_K3075000

PAM-SnoopT

Introduction

PAM-Snooptag-His 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) The SnoopTag is a small polypeptide tag that spontaneously forms an isopeptide bond between reactive amino acid side chains to its corresponding SnoopCatcher (Brune, 2017). This system opens up a variety of applications, utilising the catcher-tag conjugation system for bioconjugation and synthetic assembly of the DBAT enzyme to SnoopCatcher containing proteins.

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The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.

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.

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Characterisation

Protein expression assay

Cells containing a plasmid with the PAM insert (as confirmed by colony PCR and sequencing) were grown up and a sample of this was used to perform a protein expression assay. Bug buster was used to separate soluble and insoluble proteins.

Figure ?. Protein expression assay using bug buster to determine expression of Phenylalanine aminomutase (PAM) as soluble and insoluble form.