| 摘要 |
The present invention discloses the engineering of a human enzyme with arginine hydrolytic activity suitable for human therapy. An enzyme comprising of a human sequence is not likely to induce adverse immunological responses and thus is expected to constitute a superior therapeutic. Since the human genome does not encode arginases with the proper high affinity catalytic properties (i.e., for example, a low Km and high catalytic activity, kcat) an appropriate arginase can be engineered by modifying an enzyme with related catalytic activity. For example, the human enzyme PAD4 can hydrolyze arginine in peptide substrates but does not have activity for free arginine. First, a high throughput assay was developed for detecting arginine activity by monitoring the formation of the hydrolytic product citrulline. Then, using a combination of rational design and iterative mutation and screening PAD4 mutants were identified and isolated exhibiting high affinity free arginine metabolic activity. These mutants did not retain activity for their original substrate, peptidyl arginine.
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