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Co-expression of the subunits of the heterodimer of HIV-1 reverse transcriptase in Escherichia coli

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Müller,  Barbara
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Restle,  Tobias
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Weiss,  Stefan
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Gautel,  Mathias
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Sczakiel,  Georg
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Goody,  Roger S.
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Müller, B., Restle, T., Weiss, S., Gautel, M., Sczakiel, G., & Goody, R. S. (1989). Co-expression of the subunits of the heterodimer of HIV-1 reverse transcriptase in Escherichia coli. The Journal of Biological Chemistry, 264(24), 13975-13978. doi:10.1016/S0021-9258(18)71628-1.


Cite as: https://hdl.handle.net/21.11116/0000-000B-5EDB-6
Abstract
Expression of the 66-kDa form of human immunodeficiency virus, type 1 reverse transcriptase in Escherichia coli leads to isolation of small amounts of a 2 x 66-kDa homodimer and larger amounts of a heterodimer form of the enzyme in which the 66-kDa protein is complexed with its carboxyl-terminally truncated is complexed with its carboxyl-terminally truncated 51-kDa form. The latter arises via proteolysis by contaminating proteases. The heterodimer, which was characterized by gel filtration (apparent native molecular mass of 120-130 kDa), was the most active form of the enzyme (specific activity, 5000 units/mg, cf. less than 2000 for the 66-kDa fragment). The 66-kDa fragment alone was shown to be only partially dimerized, with the activity residing mainly in the dimer fraction. Proteolysis of the 66-kDa form resulting partially in the 51-kDa form led to an increase in reverse transcriptase activity. Expression of a truncated version of the protein containing the first 428 amino acids of the reverse transcriptase coding region led to a protein which had low but measurable reverse transcriptase activity (400-500 units/mg). Co-expression of the two proteins on a single plasmid led to expression in a 1:1 ratio. The 1:1 mixture behaved as a heterodimer, as shown by its chromatographic properties. It is likely that the mechanism for the production of heterodimers in vivo involves cleavage of 66-kDa monomers followed by rapid dimerization of the 51- and 66-kDa forms to give the heterodimeric form, which is stable toward further proteolysis.