Rapid gastric fluid digestion and biochemical characterization of engineered proteins enriched in essential amino acids

The barley high lysine (BHL) proteins are nutritionally enhanced derivatives of barley chymotrypsin inhibitor-2 (CI-2). A compactly folded new CI-2 derivative, BHL9, was engineered with the highest content of threonine, tryptophan, and isoleucine yet achieved in this protein family (15.1, 9.4, and 12.1 wt %, respectively). BHL9 had an unfolding midpoint of 5.5 M guanidinium chloride, significantly greater than values for wild type (3.9 M) or for the previously most stable BHL protein, BHL8 (3.6 M). BHL9 and all other derivatives were digested within 15 s in simulated gastric fluid (SGF), suggesting nutritional availability upon ingestion. Denaturation of the proteins in SGF minus pepsin was revealed by changes in their fluorescence emission spectra and/or far UV circular dichroism spectra. The proteins lack homology to known allergens. Significantly, the BHL8 and BHL9 proteins were stable to proteases at pH 7.5 or 8.0, attesting to their potential for high expression in plants.