Recombinant terminal deoxynucleotidyl transferase with improved functionality
AssigneeRoche Diagnostics Operations, Inc.
InventorsRainer Mueller, Markus Pajatsch, Ingo Curdt, Harald Sobek, Manfred Schmidt, Bernhard Suppmann, Kirsten Sonn, Bernd Schneidinger
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Abstract
Truncated terminal deoxynucleotidyl transferase (TdT) derivative from calf thymus, characterized in that the derivative in comparison to the native TdT is N-terminally truncated by up to 161 amino acids and has a 20- to 30-fold higher enzyme activity in solutions containing Co2+ ions, and its recombinant production and use.
Claims11
A recombinantly expressed terminal deoxynucleotidyl transferase (TdT) fusion protein comprising a truncated terminal deoxynucleotidyl transferase sequence and a protein tag sequence N-terminally fused to said truncated terminal deoxynucleotidyl transferase sequence, wherein said protein tag is selected from the group consisting of a His tag, Streptavidin binding proteins, Maltose binding proteins, GST and HA tags, and wherein the truncated terminal deoxynucleotidyl transferase sequence consists of the native calf thymus TdT sequence of SEQ ID NO:
- 2 N-terminally truncated by 138, 152, or 161 amino acids, wherein said recombinantly expressed terminal deoxynucleotidyl transferase fusion protein has a 20- to 30-fold higher enzyme activity in solutions containing Co2+ ions relative to solutions containing Zn/Mg ions.
A recombinantly expressed terminal deoxynucleotidyl transferase (TdT) fusion protein comprising a truncated terminal deoxynucleotidyl transferase sequence and a protein tag sequence N-terminally fused to said truncated terminal deoxynucleotidyl transferase sequence, wherein said protein tag is selected from the group consisting of a His tag, Streptavidin binding proteins, Maltose binding proteins, GST and HA tags, and wherein the truncated terminal deoxynucleotidyl transferase sequence consists of the native calf thymus TdT sequence of SEQ ID NO:
- 2 N-terminally truncated by 100 to 160 amino acids, wherein said recombinantly expressed terminal deoxynucleotidyl transferase fusion protein has a 20- to 30-fold higher enzyme activity in solutions containing Co2+ ions relative to solutions containing Zn/Mg ions.
A composition comprising a homogenous population of a recombinantly expressed terminal deoxynucleotidyl transferase (TdT) fusion protein, wherein the recombinantly expressed terminal deoxynucleotidyl transferase comprises a truncated terminal deoxynucleotidyl transferase sequence and a His tag sequence N-terminally fused to said truncated terminal deoxynucleotidyl transferase sequence, wherein said truncated terminal deoxynucleotidyl transferase sequence consists of the sequence of SEQ ID NO:
- 2 N-terminally truncated by at least 100 amino acids and up to 160 amino acids, wherein said recombinantly expressed terminal deoxynucleotidyl transferase fusion protein has a 20- to 30-fold higher enzyme activity in solutions containing Co2+ ions relative to solutions containing Zn/Mg ions.
Dependencies are inferred from the claim text.