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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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Staphylokinase
Staphylokinase is a protein produced by a number of strains of Staphylococcus aureus, which also displays therapeutic potenital as a thrombolytic agent. The protein has been purified from its natural source by a combination of ammonium sulphate precipitation and cation-exchange chromatography on CM cellulose. Affinity chromatography using plasmin or plasminogen immobilized to sepharose beads has also been used. The pure product is a 136 amino acid polypeptide displaying a molecular mass in the region of 16.5 kDa. Lower molecular mass derivatives lacking the first six or 10 NH2-terminal amino acids have also been characterized. All three appear to display similar thrombolytic activity in vitro at least.
The staphylokinase gene has been cloned in E. coli, as well as various other recombinant systems. The protein is expressed intracellularly in E. coli at high levels, representing 10-15% of total cellular protein. It can be purified directly from the clarified cellular homogenate by a combination of ion-exchange and hydrophobic interaction chromatography.
Although staphylokinase shows no significant homology with streptokinase, it induces a thrombolytic effect by a somewhat similar mechanism — it also forms a 1:1 stoichiometric complex with plasminogen. The proposed mechanism by which staphylokinase induces
BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 387
Staphylokinase
Free plasmin Free plasminogen
Figure 9.20. Schematic representation of the mechanism by which staphylokinase appears to activate the thrombolytic process via the generation of plasmin. See text for details
plasminogen activation is outlined in Figure 9.20. Binding of the staphylokinase to plasminogen appears to initially yield an inactive staphylokinase-plasminogen complex. However, complex formation somehow induces subsequent proteolytic cleavage of the bound plasminogen, forming plasmin, which remains complexed to the staphylokinase. This complex (via the plasmin) then appears to catalyse the conversion of free plasminogen to plasmin, and may even accelerate the process of conversion of other staphylokinase-plasminogen complexes into staphylokinase-plasmin complexes. The net effect is generation of active plasmin, which
388 BIOPHARMACEUTICALS
displays a direct thrombolytic effect by degrading clot-based fibrin, as described previously (Figure 9.18).
The serum protein a2-antiplasmin can inhibit the activated plasmin-staphylokinase complex. It appears that the a2-antiplasmin can interact with the active plasmin moiety of the complex, resulting in dissociation of staphylokinase, and consequent formation of an inactive plasmin-a2-antiplasmin complex.
The thrombolytic ability of (recombinant) staphylokinase has been evaluated in initial clinical trials, with encouraging results; 80% of patients suffering from acute myocardial infarction who received staphylokinase responded positively (10 mg staphylokinase was administered by infusion over 30min). The native molecule displays a relatively short serum half-life (6.3 min), although covalent attachment of polyethylene glycol (PEG) reduces the rate of serum clearance, hence effectively increasing the molecule’s half-life significantly. As with streptokinase, patients administered staphylokinase develop neutralizing antibodies. A number of engineered (domain-deleted) variants have been generated, which display significantly reduced immunogenicity.
a1 -Antitrypsin
The respiratory tract is protected by a number of defence mechanisms which include:
• particle removal in the nostril/nasopharynx;
• particle expulsion (e.g. by coughing);
• upward removal of substances via mucociliary transport;
• presence in the lungs of immune cells, such as alveolar macrophages;
• production/presence of soluble protective factors, including ai-antitrypsin, lysozyme, lactoferrin and interferon.
Failure/ineffective functioning of one or more of these mechanisms can impair normal respiratory function, e.g. emphysema is a condition in which the alveoli of the lungs are damaged, which compromises the lung’s capacity to exchange gases, and breathlessness often results. This condition is often promoted by smoking, respiratory infections or a deficiency in the production of serum ai-antitrypsin.
ai-Antitrypsin is a 394 amino acid, 52 kDa serum glycoprotein. It is synthesized in the liver and secreted into the blood, where it is normally present at concentrations of 2-4 g/l. It constitutes in excess of 90% of the a1-globulin fraction of blood.
The a1-antitrypsin gene is located on chromosome 14. A number of a1-antitrypsin gene variants have been described. Their gene products can be distinguished by their differential mobility upon gel electrophoresis. The normal form is termed M, while point mutations in the gene have generated two major additional forms, S and Z. These mutations results in a greatly reduced level of synthesis and secretion into the blood of the mature a1-antitrypsin. Persons inheriting two copies of the Z gene, in particular, display greatly reduced levels of serum a1-antitrypsin activity. This is often associated with the development of emphysema (particularly in smokers). The condition may be treated by the administration of purified a1-antitrypsin. This protein constitutes the major serine protease inhibitor present in blood. It is a potent inhibitor of the protease elastase, and serves to protect the lung from proteolytic damage by inhibiting neutrophil elastase. The product is administered on an ongoing basis to sufferers, who receive up to 200 g of the inhibitor each year. It is normally prepared by limited fractionation of whole human blood, although the large quantities required by patients heightens the risk of accidental transmission of blood-borne pathogens. The ai-antitrypsin gene has been expressed in a number
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