Enhancing the rumen stability of MB-1, a de novo protein with potential application in ruminant nutrition

MB-1, or Milk Bundle-1, is a de novo protein with potential application in ruminant nutrition. Its artificially high content of the essential amino acids methionine, threonine, lysine, and leucine renders it a high quality source of limiting nutrients for the animal. However, whether expressed in rumen microbes or grown in transgenic crops, MB-1 is not stable enough for its intended use. In fact, it is labile at rumen temperature and would be rapidly degraded if exposed to rumen enzymes. With the goal of enhancing the rumen stability of MB-1, three mutants were designed and analyzed.

The first mutant, MB-1-Cys, was designed to have an exposed cysteine residue and be capable of forming an intermolecular disulfide bond.

Construction of the second mutant involved the introduction of two cysteine mutations in such a way that they would be capable of forming an intramolecular disulfide bond and potentially increasing the thermal stability of the protein. For the third mutant, MB-1-Trp, the lone tyrosine in MB-1 was designed to be replaced by a tryptophan in order to improve the spectral properties of the protein and possibly increase its thermal stability. (Abstract shortened by UMI.).