High-level expression of murine terminal deoxynucleotidyl transferase in Escherichia coli grown at low temperature and overexpressing argU tRNA.
Mol Biotechnol
; 10(3): 199-208, 1998 Dec.
Article
em En
| MEDLINE
| ID: mdl-9951698
ABSTRACT
Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5'-triphosphates onto the 3'-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.
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Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
RNA de Transferência de Arginina
/
Engenharia de Proteínas
/
DNA Nucleotidilexotransferase
Limite:
Animals
Idioma:
En
Revista:
Mol Biotechnol
Ano de publicação:
1998
Tipo de documento:
Article