Development of an optimized feeding technology for dairy cows: improvement in resistance to ruminal proteases in the de novo-designed protein MB-1.
We have previously reported on MB-1, a designer protein with potential application in animal nutrition. Having a high content of selected essential amino acids, MB-1 should provide limiting nutrients for animals and promote growth and production. However, the protein was found to have marginal conformational and proteolytic stability, and, thus, strategies for stabilizing MB-1 were elaborated. We discuss the synthesis of MB-1-Cys dimer, a protein with an intermolecular disulfide bridge. This mutant was exposed to Pronase E protease preparation as well as to proteases extracted from ruminal microbes. It was found that in both cases, MB-1-Cys dimer had a better resistance to proteolytic degradation than MB-1. Denaturation and hydrophobic dye binding studies revealed that this enhanced stability was not owing to conformational stabilization, but rather to changes in surface exposure as a consequence of dimerization. In particular, it was found that binding of ANSA to MB-1-Cys dimer was comparable to that observed for native, compact, natural proteins. We discuss the implications of these results for the design of transgenic protein production systems.
Morrison
J J
JJ
Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University, Houston 77030-3303, USA.
McNally
J D
JD
Navidzadeh
A
A
Beauregard
M
M
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000821:Animal Feed; D000818:Animals; D002417:Cattle; D004044:Dietary Proteins / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D009154:Mutation; D011402:Pronase / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins; D017422:Sequence Analysis, DNA; D013696:Temperature; D013997:Time Factors
D000817:Anilino Naphthalenesulfonates; D004044:Dietary Proteins; D004220:Disulfides; C107457:MB-1synthetic dietary protein; D011506:Proteins; C039256:6-anilino-1-naphthalenesulfonic acid; D010450:Endopeptidases; D011402:Pronase