High-level expression of murine terminal deoxynucleotidyl transferase in Escherichia coli grown at low temperature and overexpressing argU tRNA

• Published in: Molecular biotechnology 1998-12, Vol.10 (3), p.199-208
• Main Authors: BOULE, J.-B, JOHNSON, E, ROUGEON, F, PAPANICOLAOU, C
• Format: Article
• Language: English
• Subjects: Animals; Bacteria; Biological and medical sciences; Biotechnology; DNA Nucleotidylexotransferase - genetics; DNA Nucleotidylexotransferase - isolation & purification; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Immunoblotting; Inclusion Bodies - chemistry; Kinetics; Low temperature; Methods. Procedures. Technologies; Mice; Modification of gene expression level; Protein Engineering - methods; Recombinant Proteins - biosynthesis; RNA, Transfer, Arg - biosynthesis; RNA, Transfer, Arg - isolation & purification; Solubility; T cell receptors; Temperature; Vertebrates
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/BF02740839

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.