Structure and function of streptokinase-microplasminogen fusion protein (CROSBI ID 467505)
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Podaci o odgovornosti
Brdar, Branko ; Čović, Marcela ; Reich, E.
engleski
Structure and function of streptokinase-microplasminogen fusion protein
We have constructed, cloned and expressed microplasminogen (mPlg), consisting of the N-terminal undecapeptide of human plasminogen (Plg) spliced to its proenzyme domain. This truncated (~28 kDa) proenzyme retained the distinctive catalytic activities of the larger parent protein. Hence, activation of mPlg occurs by limited proteolysis of the R19-V20 peptide bond in the zymogen, liberating a new N-terminal segment (VVGG-) within the two chain disulfide-linked proteolytic enzyme microplasmin (mPlm). This activation is catalysed by relevant plasminogen activators (PA), urokinase (uPA) and tissue type PA (tPA); mPlg can also be activated by streptokinase (SK), a bacterial protein (47 kDa) which forms 1:1 complexes with Plg or Plm. These complexes then act as PAs and cleave the same R-V bond that is cleaved by uPA or tPA. The role of the new N-terminus in mPlg activation with uPA or SK was probed by using protein engineering and site-directed proteolysis to alter the length and amino acid composition of the N-terminus. All modifications affected both Km and kcat. In the Plg-SK complex SK reversibly induces a typical serine protease active site that expresses characteristic PA activity in the absence of covalent modification of either Plg or SK. To determine whether the covalent modifications of Plg-SK complex affects its substrate specifity, we genetically engineered a fusion protein between SK and mPlg in both orientations. When expressed in the Pichia system, this chimeric protein was proteolitically degraded giving rise to two major products of 28 and 34 kDa. To nullify the potential for this proteolytic effect, we substituted in the SK sequence four pairs of positively charged with uncharged amino acids and fused such SK with mPlg; stability and enzymatic properties of modified SK-Plg fusion protein are currently being examined.
streptokinase; microplasminogen; protein engineering
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Podaci o prilogu
23-x.
1998.
objavljeno
Podaci o matičnoj publikaciji
Podaci o skupu
First Cogress of Croatian Geneticists with International Participation
poster
01.06.1998-04.06.1998
Hvar, Hrvatska