Abstract - Controlling proteolytic degradation of the methionine enriched MB-1Trp protein

Controlling proteolytic degradation of the methionine enriched MB-1Trp protein

Maxime Sasseville
Groupe de recherche en énergie et information biomoléculaire
Département de Chimie-Biologie
Université du Québec à Trois-Rivières
Trois-Rivières, Québec, Canada
Tel: 1 819 376 5011 #3354
Fax: 1 819 376 5084
E-mail: maxime_sasseville@uqtr.ca

Catherine St-Louis
Groupe de recherche en énergie et information biomoléculaire
Département de Chimie-Biologie
Université du Québec à Trois-Rivières
Trois-Rivières, Québec, Canada
Tel: 1 819 376 5011 #3354
Fax: 1 819 376 5084
E-mail: catherine_stlouis@yahoo.ca

Habib Khoudi
Groupe de recherche en énergie et information biomoléculaire
Département de Chimie-Biologie
Université du Québec à Trois-Rivières
Trois-Rivières, Québec, Canada
Tel: 1 819 376 5011 #3354
Fax: 1 819 376 5084
E-mail: habib_khoudi@uqtr.ca

Marc Beauregard*
Groupe de recherche en énergie et information biomoléculaire
Département de Chimie-Biologie
Université du Québec à Trois-Rivières
Trois-Rivières, Québec, Canada
Tel: 1 819 376 5011 #3354
Fax: 1 819 376 5084
E-mail: marc_beauregard@uqtr.ca

*Corresponding author

Financial support: This work was supported by grants from CORPAQ (Gouvernement du Québec) and NSERC (Canada) awarded to M.B.

Keywords: agro-biotechnology, essential amino acids, protein design, protein engineering, proteolysis.

Abbreviations:
A: alanine;
ANSA: anilinonaphthalene sulfonic acid;
BCA: bicinchoninic acid;
D: aspartic acid,
E: glutamic acid;
EAA: essential amino acids;
F: phenylalanine;
H: histidine;

K: lysine;
L: leucine;
M: methionine;
MBP: maltose-binding protein;
N: asparagine;
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis;
V: valine.

Protein design is currently used for the creation of new proteins with desirable traits, which include a superior nutritional value. One of the challenges of protein design in this area is to achieve the production of stable native-like proteins that resist the proteolytic pressure of the organism used for its production (the bioreactor). We report here the identification of a specific peptide bond sensitive to E. coli proteolysis in the designer protein MB-1Trp. In an attempt to reduce proteolysis, we have created a MB-1TrpHis gene library in which the two amino acids surrounding the peptide bond, N44 and L45, were randomized using degenerated oligonucleotides. The initial characterization of MB-1TrpHis N44E/L45V and MB-1TrpHis N44E/L45M, 2 variants of the library that were more resistant than the parent protein, was performed in order to investigate the nature of the mutants' resistance. Our results suggest that the mutants behaved like MB-1Trp regarding folding and thermal stability, and that proteolytic resistance is due to the elimination of the protease recognition site.