LARGE-SCALE EXPRESSION, PURIFICATION AND CHARACTERIZATION OF SMALL FRAGMENTS OF THROMBOMODULIN - THE ROLES OF THE 6TH-DOMAIN AND OF METHIONINE-388

Fragments of human thrombomodulin (TM) have been expressed in large qu antities in the Pichia pastoris yeast expression system and purified t o homogeneity, Fermentation of P.pastoris resulted in yields of 170 mg /l TM, Purification to homogeneity resulted in an overall 10% yield, s o that quantities of similar to 20 mg purified fragments can be readil y obtained. Smaller fragments of TM, such as the individual fourth or fifth domains, were not active, nor were equimolar mixtures of the two domains, These results demonstrate that the fourth and fifth epiderma l growth factor (EGF)-like domains together comprise the smallest acti ve fragment of TM, The fragment containing the fourth and fifth EGF-li ke domains [TMEGF(4-5)] had 10% the specific activity of rabbit TM, Co mparison of the M388L mutant TMEGF(4-5) fragment with the same mutant TMEGF(4-5-6) fragment showed that the fragment: with the sixth domain had a 10-fold better K-m value for thrombin than the fragment that did not contain the sixth domain; this factor completely accounts for the higher specific activity of the fragments containing the sixth domain . Comparison of the wild-type and M388L mutants showed that the M388L mutation resulted in a 2-fold increase in k(cat) for the activation of protein C by the thrombin-TM fragment complex, completely accounting for the 2-fold increase in specific activity of these mutant fragments .