Part:BBa_K3272003

C_LTNF_C without stop codon in pPIC9K

Short description: C_LTNF_C without stop codon in pPIC9K

Long description: The original coding sequence of the LTNF is inserted in a pPIC9K vector to express it in Pichia pastoris. In this construct, we excluded stop codon before the histidine cleavage site of the vector. By this way, we gain our protein with histidine tag, which will make easier to detect and purify our protein. An IPTG inducible lac promoter is included immediately after the prefix. The RBS J61101 proceeds LTNF10 coding sequence. This is followed by a Human Rhino Virus protease site and then Myc and 6xHIS tags on the tail end.

The DNA sequence was optimized for use in Pichia pastoris by using IDT’s Codon Optimization tool. Two Cysteine amino acids have been added as the first (1) and last (26) amino acids in the part. The two Cysteine amino acids form a disulphide bridge, thereby connecting the ends of the polypeptide chain together resulting in a more circularized structure, this process is called, “Circularization.”

Spacer: Early modeling studies indicated that placing two Cysteine amino acids right next to the LTNF (Active site) sequence caused a great deal of strain, reducing its c-score as well as its activity. Four Glycine amino acids were found to greatly reduce the stress found between the first cysteine amino acid and the LTNF 10 active site, whilst also avoiding interfering with its biochemical properties, given its chemically inert and simplistic nature. The vector contains AOX1 promoter for methanol-induced expression, α-factor secretion signal to direct secreted expression, kanamycin and ampicillin resistance gene for selectivity, the c-myc epitope and polyhistidine (6xHis) tag for detection and purification of our protein.

Extension: Adding a Cystiene bond to the end of the 10 amino acid LTNF sequence resulted in stress to the end part of the active site. As a way to combat this, we began adding the natural continuation of the LTNF 10 amino acids bit by bit, the highest c-score occurred when exactly an additional 10 amino acids (extensions) were added to the LTNF 10, striking a balance between the least stress and highest entropy levels.

The coding region has the same sequence of LTNF 2.0 coding region that we submitted last year with part number BBa_K2815001; however it is now cloned in another vector, pPIC9K instead of pPICZalphaA that we used last year.

The expression vector that we used last year, pPICZalphaA, contains zeocin as a selective marker. As we aim to manufacture our end product, we conclude that using zeocin antibiotic is not cost-effective. Instead, we used pPIC9K expression vector which contains kanamycin and ampicillin as selective markers, which are much more cheaper than zeocin. Both vectors contain AOX1 promoter enabling methanol-induced expression; and α-factor secretion signal to direct secreted expression of our protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]