Physico-chemical properties of recombinant desulphatohirudin.
Physico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies.
Division of Biotechnology, PHLS Centre for Applied Microbiology & Research, Salisbury, England.
A
A
Electricwala
L
L
Irons
R
R
Wait
R J
RJ
Carr
R J
RJ
Ling
H
H
Grossenbacher
T
T
Atkinson
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D005343:Fibrinolytic Agents; D006629:Hirudins / Q000031:analogs & derivatives*; D013058:Mass Spectrometry / Q000379:methods; D011994:Recombinant Proteins; D013057:Spectrum Analysis / Q000379:methods
D005343:Fibrinolytic Agents; D006629:Hirudins; D011994:Recombinant Proteins; C060225:desirudin