Co-expression of the Subunits of the Heterodimer of HIV-1 Reverse Transcriptase in Escherichia coli

B Müller,T Restle,S Weiss,M Gautel,G Sczakiel,R S Goody

doi:10.1016/s0021-9258(18)71628-1

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.

Highlights

From the Abteilung Biophysik, Man-Planck Institut fur Medizinische Forschung, Jahnstrasse 29 and the SDeutsches Krebsforschungszentrum, Im Neuenkimer Feld 280, 6900 Heidelberg, Federal Republic of Germany obvious recognition sequence for the viral protease(typically a n aromatic residue followed by a proline,althoughquite different sequences are cleaved (Darke et aL., 1988))
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
Proteolysis of the 66-kDa form resulting partially in the 51-kDa form led to an increase in reverse transequenceand todelete the endonuclease sequence downstream of the RT coding region and introducinga TAA-stop codon simultaneously the site-directemdutagenesis method described by Taylor et al (1985) was used

Summary

Introduction

From the Abteilung Biophysik, Man-Planck Institut fur Medizinische Forschung, Jahnstrasse 29 and the SDeutsches Krebsforschungszentrum, Im Neuenkimer Feld 280, 6900 Heidelberg, Federal Republic of Germany obvious recognition sequence for the viral protease(typically a n aromatic residue followed by a proline,althoughquite different sequences are cleaved (Darke et aL., 1988)). The heterodimer, whichwas to create an EcoRI restriction site at the 5’ end of the RT coding characterized by gel filtration(apparent native molecular mass of 120-130 kDa), wasthe most active form of the enzyme (specificactivity, 5000 units/mg, cf e2000 for the 66-kDa fragment).

Results

Conclusion