Novel method of expression and purification of hirudin based on pBAD TOPO, pTYB12 vectors and gene synthesis.
To express recombinant hirudins in Escherichia coli cells, a fragment of chemically synthesized DNA was used, containing codons for the individual amino acids preferred by the host cells. Gene synthesis was based on the design of two DNA fragments, so-called mega primers H1 and H2 with a complementary fragment, and their incubation with Taq polymerase. The gene obtained in this fashion was multiplied using the PCR, and then expressed in E. coli cells with the use of TOPO vectors pBAD and pTYB12. Using this method, hirudins were obtained in the amount of 17 mg/l E. coli strain, with the activity of 17 antithrombin units (ATU)/mg protein. The method can be considered as an easy and inexpensive route to small protein synthesis.
Kochanowski
Rafał
R
Department of Food Chemistry, Technology and Biotechnology, Gdansk University of Technology, Chemical Faculty, Narutowicza St. 11/12, 80-952 Gdansk, Poland. rkoch@chem.pg.gda.pl
Kotłowski
Roman
R
Szweda
Piotr
P
D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006629:Hirudins / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013917:Thrombin / Q000187:drug effects
D017931:DNA Primers; D006629:Hirudins; D011994:Recombinant Proteins; D004247:DNA; D013917:Thrombin