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Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems
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| Zeitschriftentitel: | European Journal of Biochemistry |
|---|---|
| Personen und Körperschaften: | , , , , , |
| In: | European Journal of Biochemistry, 234, 1995, 3, S. 811-818 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Wiley
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| Schlagwörter: |
| author_facet |
Pekrun, Katja Petry, Harald Jentsch, Klaus‐Dieter Moosmayer, Dieter Hunsmann, Gerhard Lüke, Wolfgang Pekrun, Katja Petry, Harald Jentsch, Klaus‐Dieter Moosmayer, Dieter Hunsmann, Gerhard Lüke, Wolfgang |
|---|---|
| author |
Pekrun, Katja Petry, Harald Jentsch, Klaus‐Dieter Moosmayer, Dieter Hunsmann, Gerhard Lüke, Wolfgang |
| spellingShingle |
Pekrun, Katja Petry, Harald Jentsch, Klaus‐Dieter Moosmayer, Dieter Hunsmann, Gerhard Lüke, Wolfgang European Journal of Biochemistry Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems Biochemistry |
| author_sort |
pekrun, katja |
| spelling |
Pekrun, Katja Petry, Harald Jentsch, Klaus‐Dieter Moosmayer, Dieter Hunsmann, Gerhard Lüke, Wolfgang 0014-2956 1432-1033 Wiley Biochemistry http://dx.doi.org/10.1111/j.1432-1033.1995.811_a.x <jats:p>To produce the human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV‐1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases.</jats:p><jats:p>The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as B p66/p60 heterodimer. The recombinant His‐RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N‐terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and <jats:italic>in vitro</jats:italic> activation by viral and non‐viral proteases. The recombinant His‐RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an <jats:italic>Escherichia coli</jats:italic> ‐expressed RT. Removal of the hexahistidine tag from the recombinant His‐RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His‐RT.</jats:p> Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems European Journal of Biochemistry |
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10.1111/j.1432-1033.1995.811_a.x |
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Wiley, 1995 |
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European Journal of Biochemistry |
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49 |
| title |
Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_unstemmed |
Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_full |
Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_fullStr |
Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_full_unstemmed |
Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_short |
Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_sort |
expression and characterization of the reverse transcriptase enzyme from type 1 human immunodeficiency virus using different baculoviral vector systems |
| topic |
Biochemistry |
| url |
http://dx.doi.org/10.1111/j.1432-1033.1995.811_a.x |
| publishDate |
1995 |
| physical |
811-818 |
| description |
<jats:p>To produce the human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV‐1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases.</jats:p><jats:p>The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as B p66/p60 heterodimer. The recombinant His‐RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N‐terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and <jats:italic>in vitro</jats:italic> activation by viral and non‐viral proteases. The recombinant His‐RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an <jats:italic>Escherichia coli</jats:italic> ‐expressed RT. Removal of the hexahistidine tag from the recombinant His‐RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His‐RT.</jats:p> |
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| author | Pekrun, Katja, Petry, Harald, Jentsch, Klaus‐Dieter, Moosmayer, Dieter, Hunsmann, Gerhard, Lüke, Wolfgang |
| author_facet | Pekrun, Katja, Petry, Harald, Jentsch, Klaus‐Dieter, Moosmayer, Dieter, Hunsmann, Gerhard, Lüke, Wolfgang, Pekrun, Katja, Petry, Harald, Jentsch, Klaus‐Dieter, Moosmayer, Dieter, Hunsmann, Gerhard, Lüke, Wolfgang |
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| container_title | European Journal of Biochemistry |
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| description | <jats:p>To produce the human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV‐1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases.</jats:p><jats:p>The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as B p66/p60 heterodimer. The recombinant His‐RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N‐terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and <jats:italic>in vitro</jats:italic> activation by viral and non‐viral proteases. The recombinant His‐RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an <jats:italic>Escherichia coli</jats:italic> ‐expressed RT. Removal of the hexahistidine tag from the recombinant His‐RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His‐RT.</jats:p> |
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| imprint | Wiley, 1995 |
| imprint_str_mv | Wiley, 1995 |
| institution | DE-105, DE-14, DE-Ch1, DE-L229, DE-D275, DE-Bn3, DE-Brt1, DE-Zwi2, DE-D161, DE-Gla1, DE-Zi4, DE-15, DE-Rs1, DE-Pl11 |
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| physical | 811-818 |
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| spelling | Pekrun, Katja Petry, Harald Jentsch, Klaus‐Dieter Moosmayer, Dieter Hunsmann, Gerhard Lüke, Wolfgang 0014-2956 1432-1033 Wiley Biochemistry http://dx.doi.org/10.1111/j.1432-1033.1995.811_a.x <jats:p>To produce the human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV‐1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases.</jats:p><jats:p>The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as B p66/p60 heterodimer. The recombinant His‐RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N‐terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and <jats:italic>in vitro</jats:italic> activation by viral and non‐viral proteases. The recombinant His‐RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an <jats:italic>Escherichia coli</jats:italic> ‐expressed RT. Removal of the hexahistidine tag from the recombinant His‐RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His‐RT.</jats:p> Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems European Journal of Biochemistry |
| spellingShingle | Pekrun, Katja, Petry, Harald, Jentsch, Klaus‐Dieter, Moosmayer, Dieter, Hunsmann, Gerhard, Lüke, Wolfgang, European Journal of Biochemistry, Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems, Biochemistry |
| title | Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_full | Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_fullStr | Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_full_unstemmed | Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_short | Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| title_sort | expression and characterization of the reverse transcriptase enzyme from type 1 human immunodeficiency virus using different baculoviral vector systems |
| title_unstemmed | Expression and Characterization of the Reverse Transcriptase Enzyme from Type 1 human Immunodeficiency Virus Using Different Baculoviral Vector Systems |
| topic | Biochemistry |
| url | http://dx.doi.org/10.1111/j.1432-1033.1995.811_a.x |