Liao H H
University of Wisconsin Biotechnology Center, Madison 53705.
Protein Expr Purif. 1991 Feb;2(1):43-50. doi: 10.1016/1046-5928(91)90008-7.
The expression of kanamycin nucleotidyltransferase (KNTase) in Escherichia coli results in different forms of the protein, depending on the temperature; soluble active enzyme is synthesized at 23 degrees C but the protein is mostly aggregated and inactive in inclusion bodies when made at 37 degrees C. However, active enzyme can be recovered by solubilization of the inclusion bodies with 8 M urea followed by dilution of the denaturant, indicating that the polypeptide is not damaged covalently but is present in a misfolded state. The availability of thermostable mutants of KNTase allows a test of the hypothesis that formation of inclusion bodies when proteins are highly expressed in E. coli is due to the accumulation of a folding intermediate that is prone to temperature-dependent aggregation. Because these mutants were isolated by cloning the KNTase gene into the thermophile Bacillus stearothermophilus and selecting for kanamycin resistance at high growth temperatures, they must be thermostable for both synthesis and activity and must have folding intermediates that are less susceptible to the formation of aggregates. Indeed, whereas decreasing the temperature from 37 to 23 degrees C increased the KNTase specific activity 10-fold in cells expressing the wild-type enzyme, this change resulted in only a 2.1-fold increase for the TK1 (Asp80----Tyr) mutant and a 1.7-fold increase for the TK101 (Asp80----Tyr and Thr130----Lys) double mutant. The strategy of cloning in thermophiles and selecting or screening for mutants that fold correctly to yield biological activity at high growth temperatures may be useful in overcoming the problem of the insolubility of some proteins when expressed in heterologous hosts.
卡那霉素核苷酸转移酶（KNTase）在大肠杆菌中的表达会产生不同形式的蛋白质，这取决于温度；在23℃时合成的是可溶性活性酶，但在37℃时产生的蛋白质大多聚集在包涵体中且无活性。然而，通过用8M尿素溶解包涵体，然后稀释变性剂，可以回收活性酶，这表明多肽没有共价损伤，而是以错误折叠的状态存在。KNTase热稳定突变体的可得性使得可以检验这样一个假设：当蛋白质在大肠杆菌中高表达时形成包涵体是由于易于温度依赖性聚集的折叠中间体的积累。由于这些突变体是通过将KNTase基因克隆到嗜热脂肪芽孢杆菌中并在高生长温度下选择卡那霉素抗性而分离得到的，它们在合成和活性方面都必须是热稳定的，并且必须具有不易形成聚集体的折叠中间体。实际上，在表达野生型酶的细胞中，将温度从37℃降至23℃会使KNTase比活性提高10倍，而对于TK1（Asp80→Tyr）突变体，这种变化仅导致比活性提高2.1倍，对于TK101（Asp80→Tyr和Thr130→Lys）双突变体，比活性提高1.7倍。在嗜热菌中克隆并选择或筛选能够正确折叠以在高生长温度下产生生物活性的突变体的策略，可能有助于克服某些蛋白质在异源宿主中表达时的不溶性问题。
