Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388.
Fragments of human thrombomodulin (TM) have been expressed in large quantities in the Pichia pastoris yeast expression system and purified to homogeneity. Fermentation of P. pastoris resulted in yields of 170 mg/l TM. Purification to homogeneity resulted in an overall 10% yield, so that quantities of approximately 20 mg purified fragments can be readily 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 epidermal growth factor (EGF)-like domains together comprise the smallest active fragment of TM. The fragment containing the fourth and fifth EGF-like domains [TMEGF(4-5)] had 10% the specific activity of rabbit TM. Comparison 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 Km 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 kcat 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.
White
C E
CE
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0601, USA.
Hunter
M J
MJ
Meininger
D P
DP
White
L R
LR
Komives
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004815:Epidermal Growth Factor; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology*; D010843:Pichia / Q000235:genetics; D011486:Protein C / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D018180:Thrombomodulin / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D004815:Epidermal Growth Factor