Part:BBa_K3075003

LXYL-P1-2- SpyT

Introduction

LXYL-P1-2- SpyT consists of the enzyme Beta-D-xylosidase fused at the C-terminus to a short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of LXYL originated from Lentinula edodes (Shiitake mushroom).(1)

Usage and Biology

In Lentinula edodes, Beta-D-xylosidase (LXYL) catalyses the hydrolysis of O-glycosyl bonds. LXYL has also been used to hydrolyse the β-xylosyl group of 7-β-Xylosyl-10-deacetyltaxol (XDT) to form 10-deacetyltaxol (DT) in an alternate pathway of paclitaxel biosynthesis. Recombinant LXYL has a sequence of 803 amino acid residues with a molecular mass of 85,975 Da.

Characterisation

The LXYL-P1-2- SpyT gBlock was synthesised by IDT. LXYL-SpyT 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.