Part:BBa_K2331004

Periplasmic-Localizing Endoglucanase with HIS-Tag

Endoglucanase is an enzyme which cleaves internal beta-1,4 glycosidic bonds in cellulose polymers. This part has been optimized to express a C-terminal his-tag for confirmation of expression and a N-terminal PelB sequence for localization to the periplasm for secretion or diffusion. The original part can be found here: https://parts.igem.org/Part:BBa_K2160000. Our interest in this part was to cleave cellulose polymers to dimers for downstream cleavage by beta-glucosidase (BBa_K2331003).

Proof of Expression

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This is a western blot probed with anti-HIS tag showing three species.Lane 1 is the pET26B(+) empty vector, Lane 2 is the pet26B(+) with the optimized endoglucanase. The 46 kDa species in Lane 2 is the optimized endoglucanase enzyme. The ~30 kDa species is, likely, a product initiated from an internal methionine codon that has an imperfect ribosomal binding sequence 5-10 bp upstream, resulting in some expression of this truncated species.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 87
  Illegal XhoI site found at 1153
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 54
  Illegal AgeI site found at 308
  
• 1000
  COMPATIBLE WITH RFC[1000]