Part:BBa_K3075001

DBAT^G38R/F301V-SnoopT-His

Introduction

DBAT-G38R/F301V-SnoopT consists of the enzyme 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) 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 DBAT which was used, originated from Taxus cuspidata (Japanese yew), but contained a double mutation of G38R/F301V. (2)

Usage and Biology

This protein naturally participates in the synthesis of baccatin III from 10-deacetyl-2-debenzoylbaccatin III, where it catalyses the final acetylation of. Baccatin III synthesis is a subpathway of paclitaxel biosynthesis, which is itself part of Alkaloid biosynthesis. The mutant however, has been designed to catalyse the acetylation of 10-deacetyltaxol (DT) with a catalytic efficiency approximately six times higher than that of the wild-type. (2) The recombinant mutant enzyme has a length of 440 amino acid residues, a molecular weight of 49,052 Da and an optimum pH of 7.5. (3)

Characterisation

The DBAT-G38R/F301V-SnoopT gBlock was synthesised by IDT. DBAT-G38R/F301V-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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 771
  Illegal PstI site found at 826
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 771
  Illegal PstI site found at 826
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 28
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 771
  Illegal PstI site found at 826
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 771
  Illegal PstI site found at 826
  
• 1000
  COMPATIBLE WITH RFC[1000]