Abstract
Overproduction of human terminal transferase protein has now been accomplished by cloning the coding sequence of human terminal transferase into a baculovirus, where the expression of terminal transferase is under the control of the polyhedrin protein promoter. Two constructs were made, one producing a protein containing the entire terminal transferase fused to 12 amino acids from the NH2 terminus of the polyhedrin protein, and the other producing 58-kDa human terminal transferase. The terminal transferase levels expressed in cells infected with either recombinant baculovirus are around 10,000 units/10(7) cells at 48 h postinfection, about 200-fold greater than levels expressed in thymus and cultured lymphoblastoid cells. The chimeric polyhedrin/human terminal transferase protein produced in the infected insect cells has a molecular weight of about 60,000 while the nonfused recombinant human terminal transferase is identical in molecular weight to that present in human lymphoblastoid cells. Both forms of recombinant terminal transferase show immunological and enzymatic activity. When infected cells are pulse-labeled with [35S] methionine at 42-45 h postinfection, about 10% of newly synthesized protein is terminal transferase. Both forms of terminal transferase are phosphorylated in recombinant virus-infected cells as demonstrated by pulse-labeling infected cells with 32P-inorganic phosphate and isolation of labeled terminal transferase peptides by immunoprecipitation.
Highlights
Sequence of human terminal transferase into a bacu- TdT from calf thymus glands was purified to homogeneity lovirus, where the expression of terminal transferase in 1971 as a proteolyzed form containing two peptides [9]
Cloning of the coding sequence of recombinant human terminal transferase is identical human TdT cDNA in a bacterialplasmid allowed the producin molecularweight to that present inhuman lympho- tion of enzymatically active human enzyme in Escherichia coli blastoid cells
In thispaper we describe methionine at 42-45 hpostinfection,about 10%of a useful process for overproduction of human TdT protein in newly synthesizedprotein is terminaltransferase. the baculovirus system
Summary
Terminal transferase (TdT)’ activity was first described in 1960 ( 1 ) as a side reaction present in partially purified DNA polymerase preparations from calf thymus glands. Cells indicated that the AcNPV373lTdT327-infectedculture contained 27% T d T positive cellsa t 24 h after infection while the AcNPV360/TdT331-infectedculture contained 15%T d T positive cells, the enzyme activity levels presentare comparable. This observationsuggests that the chimeric protein is produced moreeffectively than the recombinant TdT without anyleading polyhedrin amino acid sequence. The nature of the immunoreactive T d T peptides produced by the infected cells was examined by subjecting recombinant virus-infected cell lysates to analysis by SDS-PAGE andimmunoblotting.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
