Müller B, Restle T, Weiss S, Gautel M, Sczakiel G, Goody R S
Abteilung Biophysik, Max-Planck Institut für Medizinische Forschung, Heidelberg, Federal Republic of Germany.
J Biol Chem. 1989 Aug 25;264(24):13975-8.
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.
1型人类免疫缺陷病毒66 kDa形式的逆转录酶在大肠杆菌中的表达导致分离出少量的2×66 kDa同型二聚体和大量的该酶的异源二聚体形式，其中66 kDa蛋白与其羧基末端截短的51 kDa形式复合。后者是由污染的蛋白酶进行蛋白水解产生的。通过凝胶过滤表征的异源二聚体（表观天然分子量为120 - 130 kDa）是该酶最具活性的形式（比活性为5000单位/毫克，相比之下，66 kDa片段的比活性小于2000单位/毫克）。单独的66 kDa片段显示仅部分二聚化，活性主要存在于二聚体部分。66 kDa形式部分水解产生51 kDa形式导致逆转录酶活性增加。表达包含逆转录酶编码区前428个氨基酸的截短版本的蛋白质产生了一种具有低但可测量的逆转录酶活性（400 - 500单位/毫克）的蛋白质。在单个质粒上共表达这两种蛋白质导致以1:1的比例表达。如色谱特性所示，1:1混合物表现为异源二聚体。体内产生异源二聚体的机制可能涉及66 kDa单体的切割，随后51 kDa和66 kDa形式快速二聚化形成异源二聚体形式，该形式对进一步的蛋白水解稳定。
