Amino acids supplements are very popular among cattle and poultry producers because they contribute to optimising animal performances such as growth or milk protein production. These additives can overcome feed deficiencies, such as the lack of some nutritionally important amino acids known as "essential" or "limiting" amino acids in feedstock. Balancing animal diet may also be achieved by a different approach, currently under development in a number of major agro-biotechnology companies.  Such an approach focuses on the balancing of EAAs (essential amino acids) directly in the crops, which would allow for improved digestibility of these EAAs and substantial savings to the farmers.

One of the simplest ways of controlling EAA content of crops is to control the quality of the proteins being synthesized and stored in crop compartments (leaves, seeds, etc). In this regard, our group focuses on the improvement of the EAA contents of proteins. In order to achieve full control on their EAA content, the protein sequences (natural blue-print) are designed "from scratch" (de novo designed). A few years ago, one of our novel proteins called MB-1Trp was reported and shown to be very promising for agro-biotechnology application (Gagnon et al., 2000). It was not as resistant to degradation as required for its accumulation and storage in crops, and an investigation had to be undertaken in order to provide a solution to this weakness.

Degradation fragments of MB-1Trp were analysed and the exact location of cleavage was identified. Analysis of the fragments indicated that the bond between amino acid residue 44 and 45 was the most susceptible to protease attack.  Using random mutagenesis (which promoted modifications in our protein's gene and a consequent modification of the amino acids in positions 44 and 45), a small group of MB-1Trp variants was generated and analysed. The replacement of asparagine 44 by a glutamate residue, accompanied by a replacement of leucine 45 by a valine residue, led to an important improvement of resistance to degradation (other combinations did result in similar improvement, Figure 1). Such a high level of resistance maximises the probability that the protein be stored in large quantities in crop tissues.

The kind of strategy used in this project is predicted to have little impact on the digestibility of MB-1Trp in a given animal digestive tract, which obviously is an important parameter. There, diet proteins are exposed to low pH which can overcome the non-covalent forces that maintain MB-1Trp folded and expose its skeleton to digestive enzymes.

GAGNON, Mylène Claude; WILLIAMS, Martin; DOUCET, Alain and BEAUREGARD, Marc. Replacement of tyr62 by trp in the designer protein milk bundle-1 results in significant improvement of conformational stability. FEBS Letters, November 2000, vol. 484, no. 2, p. 144-148.

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