- The Case of Recombinant Streptokinase-Hazards in the Use of Synthetic Substrates for In Vitro Assays for Therapeutic Enzymes

Some ago I posted some thoughts on the in vitro assays for therapeutic enzymes with the focus on the perils of using a synthetic substrate instead of a substrate approximating the physiological substrate.   Another example of this problem is provided by the recent work of Thelwell and Longstraff (1) on assay of recombinant streptokinase.   This is clever piece of work that should be mandatory for all involved in the manufacture of recombinant proteins. 

Thelwell and Longstaff (1) observed that there was a discrepancy in streptokinase activity measured with  a chromogenic substrate (D-Val-Leu-Lys-p-nitroanilide) and activity measured by activation of plasminogen in fibrin clot assay (2).  It was shown that low activity n the fibrin clot assay was due to the presence of an N-terminal methionine which had not removed during expression in the E.coli system (3,4). Enzymatic removal of the terminal methionine resulted in increased rates of clot lysis.  Clearly the presence of the N-terminal methionine would result in a product with decreased biological activity although this defect would not be demonstrated by the use of a synthetic substrate.

1.  Thelwell, C. and Longstaff, C., Biosimilars: the process in the product.  The example of recombinant streptokinase, J.Thromb.Haemost. 12, 1229-1233, 2014.
2.  Longstaff, C. and Whtten, C.M., A proposed reference method for plasminogen activation that enable calculation of enzyme activity in SI units, J.Thromb.Haemost. 2, 1416-1421, 2004.
3.  Hirel, P., Schmitter, M.J., Dessen , P., et al., Extent of N-terminal methionne excision from Escherichia coli protein is governed by the side-chain length of the penulimate amino acid, Proc.Natl.Acad.Sci. USA 86, 8247-8251, 1989.
4.  Bonissone, S., Gupta, N., Romine, M., et al., N-terminal protein processing: a comparative protogenomic analysis, Mol.Cell.Proteomics 12, 14-28, 2013.