Part:BBa_K2924042

AOX promoter + Lactoferrin + HisTag + AOX Terminator

The heterologous expression of proteins has an important industrial role, e.g. for the production of insulin¹. Identification of new peptide and protein pharmaceuticals and the optimization of the expression of known pharmaceuticals represent a huge research sector. Around 155 pharmaceuticals and vaccines developed by bio-pharmaceutical companies were approved by the US Food and Drug Administration in 2002, the amount of approved recombinant proteins quickly rose to over 200 in 2009². Besides insulin, medically important proteins like albumin and the human growth hormone (HGH) are produced by microbes or higher organisms. Small proteins are usually expressed in prokaryotic organisms like Escherichia coli, which enables easy, quick and cheap protein expression³. Disadvantages are the lack of post-translational modification and glycosylation, the difficult expression of large proteins and proteins with disulphide bonds^3,4. These drawbacks can be compensated by using eukaryotic yeast as an expression host. Two of the primarily used yeast expression systems are Saccharomyces cerevisiae and Pichia pastoris ⁵.

In P. pastoris, the AOX1 promoter is methanol inducible and therefore the AOX is highly expressed in the presence of methanol²². This leads to high recombinant protein yields of genes introduced into P. pastoris under influence of the AOX1 promoter²³. The pPICZB Vector from the EasySelect™ Pichia Expression Kit from Thermofisher Scientific was used, which contains an inducible AOX1 promoter and a Zeocin™ resistance gene.

The lactoferrin coding sequence originates from the organism Bos Taurus and was synthesized commercially. In addition, a NotI and an Eco72I interface were placed at both ends of the gene to clone the gene into the pPICZB vector (Fig. 1).

Fig. 1: The genetic organisation of the P. pastoris expression system with the AOX Promoter BBa_K2924039 followed by the target gene and C-terminally fused to a His-tag and AOX1 Transcription Terminator BBa_K2924040. The vector is pPICZB. E: Eco72I. N: NotI

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 1
  Illegal BamHI site found at 2232
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1220
  Illegal BsaI.rc site found at 2215