Production of the HV1 variant of hirudin by recombinant DNA methodology.
Recombinant DNA technologies now allow the preparation of virtually any polypeptide sequence. Very efficient expression systems for prokaryotic and eukaryotic cells have been developed which may yield large quantities of the desired protein. Bacterial systems are still the most widely used while alternative organisms are often considered when post-translational modifications could influence the biological behaviour of the product. For hirudin or its analogues, two important molecular characteristics should be taken into account. First, it is necessary that no extra amino acid residue, such as the initial methionine, is present on the NH2 end of the recombinant polypeptide. It is known that a free N-terminal sequence is crucial for the thrombin inhibitory activity. Second, a sulphate group on tyrosine at position 63 is found in natural hirudin extracted from leeches. Such post-translational modification has never been observed for all the recombinant hirudin preparations reported to date even though the importance of the sulphate group on the in vitro and in vivo activity of hirudin has not yet been clarified. Finally, the recombinant DNA methodology of choice for the commercial development of hirudin must also take into consideration yield and cost factors which ultimately will affect the widespread use of this product particularly if it has to compete with heparin. We will review our work on the preparation of recombinant hirudin describing bacterial and insect cell expression systems and addressing some of the questions mentioned above.
de Taxis du Poet
P
P
Farmitalia Carlo Erba, Department of Biotechnology, Milan, Italy.
Scacheri
E
E
Benatti
L
L
Nitti
G
G
Valsasina
B
B
Sarmientos
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006629:Hirudins / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009036:Moths; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins; D014443:Tyrosine / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D006629:Hirudins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C072298:hirudin HV1; D014443:Tyrosine; C060225:desirudin