Part:BBa_K3075002

TycA^S563A-SpyT-His

Introduction

TycA-SpyTag-His consists of the enzyme Tyrocidine synthase 1 fused at the C-terminus to a short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. Our team utilised a sequence originating from Brevibacillus parabrevis with a S563A amino acid mutation. (1) (2)

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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

Tyrocidine Synthase 1 (TycA) is known to be involved in tyrocidine biosynthesis, a part of antibiotic biosynthesis. In the biosynthesis of the Paclitaxel side chain, TycA combines the beta-phenylalanine and the CoA thioester, and can also catalyse the production of phenylisoserinyl-CoA. (3) Recombinant TycA has a sequence of 1088 amino acid residues with a molecular mass of 122,672. (2)

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Characterisation

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Plans to characterise the TycA mutant enzyme involved measuring the loss of -SH compounds, utilising the Ellman’s assay. DTNB molecule is a compound reacts with thioester containing compounds,

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2289
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2299
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1223
  Illegal BsaI.rc site found at 2143
  Illegal SapI.rc site found at 2499