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  • Title: The position of the structurally autonomous kringle 2 domain influences the functional features of tissue-type plasminogen activator.
    Author: Bakker AH, Rehberg EF, Marotti KR, Verheijen JH.
    Journal: Protein Eng; 1995 Mar; 8(3):293-300. PubMed ID: 7479691.
    Abstract:
    Tissue-type plasminogen activator (t-PA) is composed of structurally autonomous domains. From the N-terminus of t-PA, a finger-like domain (F), an epidermal growth factor-like domain (G), two kringle domains (K1 and K2) and a serine protease domain (P) can be discerned. The K2 domain of t-PA is known to be involved in lysine binding, fibrin binding and fibrin-dependent plasminogen activation. To study the functional autonomy of the K2 domain in t-PA we constructed, with the aid of a cassette t-PA gene [Rehberg et al. (1989) Protein Engng, 2, 371-377], mutant t-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2P and GK2K1P) in which the K2 domain was placed in two different positions in t-PA. The DNAs of wild-type t-PA and the t-PA variants were expressed in Chinese hamster ovary cells and the recombinant proteins were purified by affinity chromatography. All molecules were expressed in their single-chain form and could be converted to their two-chain form. With these molecules, lysine binding, fibrin binding and fibrin-dependent plasminogen activation were studied. All variants showed affinity for lysyl-Sepharose and aminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versus FGK2K1P and GK1K2P versus GK2K1P) resulted in a 23-47% weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose. Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versus GK2K1P) resulted in a 20-70% improvement of the interactions lysyl-Sepharose and aminohexyl-Sepharose.(ABSTRACT TRUNCATED AT 250 WORDS)
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