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Expression and characterization of small fragments of thrombomodulin

Chris White

Appointment Period: 1994-1996, Grant Years: [10,11]

Chris WhiteThrombomodulin (TM) is an important anticoagulant cofactor found on the vascular endothelium. The portion of TM that is important for its cofactor activity is comprised of EGF-like domains. Active fragments of human (TM) have been expressed in large quantities in Pichia pastoris, and purified to homogeneity. The fragment containing the fourth and fifth EGF-like domains (TMEGF(4-5)) had 10% of 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 tenfold lower Km for thrombin and this difference completely accounts for the higher specific activity of fragments containing the sixth domain. Comparison of the wild type and M388L mutants showed that the M388L mutation results in a two-fold increase in Kcat for the activation of protein C by the thrombin-TM fragment complex, completely accounting for the two-fold increase in specific activity of these mutant fragments.

The disulfide bonding pattern of the fourth and fifth EGF-like domains of TM have been determined. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation released the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2carboxyethyl)phosphine. The disulfide bonding pattern of the fourth EGF-like domain was (1-3,2-4,5-6), which is the same as that found in epidermal growth factor. The disulfide bonding pattern of the fifth domain was (1-2,3-4,5-6), which is unlike that found in epidermal growth factor or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2,3-4,5-6) isomer of the fifth domain bound to thrombin more tightly than the EGF-like (1-3,2-4,5-6) isomer. The fact that not all EGF-like domains have the same disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.

PUBLICATIONS (resulting from this training)

White CE, Kempi NM, Komives EA. (1994) Expression of highly disulfide-bonded proteins in Pichia pastoris. Structure. 2:1003-5.

White CE, Hunter MJ, Meininger DP, White LR, Komives EA. (1995) Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388. Protein Eng. 8:1177-87.

White CE, Hunter MJ, Meininger DP, Garrod S, Komives EA. (1996) The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern. Proc Natl Acad Sci USA. 93:10177-82.

Vindigni A, White CE, Komives EA, Di Cera E. (1997) Energetics of thrombin-thrombomodulin interaction. Biochemistry 36:6674-81.

Gleeson MA, White CE, Meininger DP, Komives EA. (1998) Generation of protease-deficient strains and their use in heterologous protein expression. Methods Mol Biol. 103:81-94.