Display of functional thrombin inhibitor hirudin on the surface of phage M13.
A synthetic gene for hirudin was ligated into phagemid pCANTAB5E. This construct allows production of either soluble hirudin or phage having hirudin displayed on the surface. Similarly, hirudin variants with extensions either at their N- or C-terminus were generated. The genes were expressed in their soluble form in a non-suppressor strain of E. coli. Periplasmatic fractions were evaluated in standard thrombin inhibition assays. Extending hirudin by a single Gln residue at the N-terminus reduces the activity by two orders of magnitude. This suggests that either the terminal amine group makes an important interaction or that steric constraints do not allow additional amino acids here. Only C-terminal extensions maintain most of the thrombin inhibitor activity of r-hirudin. The r-hirudin gene was also expressed on the tips of filamentous phage as a fusion protein with protein III (pIII). The hirudin-pIII fusion protein was detected with anti-hirudin antibody and with anti-E-tag antibody by Western blot analysis. Recombinant phages were shown to bind to immobilized thrombin in a dose-dependent manner. Upon addition of soluble thrombin, recombinant hirudin phages could be eluted specifically. Finally, purified phages carrying displayed r-hirudin were shown to inhibit thrombin in a standard amidolytic assay for thrombin inhibitor activity. These results demonstrate that hirudin can be C-terminally extended without diminishing the antithrombic activity. Beyond that, active hirudin can be displayed on the surface of M13 phage. As a conclusion, applied molecular evolution, i.e. the selection of hirudin-based thrombin inhibitor variants with tailored properties from (partially) randomized peptide pools should now be possible.
Wirsching
F
F
Institut für Molekulare Biotechnologie, Department of Molecular Evolution Biology, Jena, Germany.
Opitz
T
T
Dietrich
R
R
Schwienhorst
A
A
D017104:Bacteriophage M13 / Q000235:genetics* / Q000378:metabolism; D002213:Capsid / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D006629:Hirudins / Q000235:genetics* / Q000378:metabolism; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors*
D006629:Hirudins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D013917:Thrombin