Part:BBa_M36850:Design

We are designing a HeLa cell actuator that produces Type I antifreeze protein. The antifreeze protein we chose is naturally found in winter flounder serum and is a Type I AFP, known as AFP9. This amino acid sequence was published in a paper by Chao in 1996. It is very short, consisting of only 52 amino acids, in which there are three repeats of the same 9-amino acid section. The amino acid content is very Alanine-rich.

Our designed part is a patchwork of components all chosen to maximize the output of our antifreeze protein. The antifreeze protein sequence starts with DNA2.0 Gene Designer software’s template kozak sequence, which is strong and promotes ribosome binding well. The kozak is then followed by the start codon, ATG, and the optimized antifreeze protein sequence. The protein sequence is followed by the stop codon TAG.

The amino acid sequence for this protein was published in the paper "A natural variant of type I antifreeze protein with four ice-binding repeats is a particularly potent antifreeze", by Heman Chao, et al. in Protein Science, Vol 5, Issue 6, pages 1150–1156 in June 1996. We optimized the nucleotides for expression in human cells.

Design Notes

We used the OPTIMIZER software with standard human codons at a guided random mix to generate the DNA sequence for the amino acid, then further manually modified some of the codons to eliminate repeated sequences.

References

1. Chao, Heman, et al. “A natural variant of type I antifreeze protein with four ice-binding repeats is a particularly potent antifreeze”. Protein Science. Vol 5, Issue 6, p1150–1156. June 1996.