title: Effect of temperature on the expression of wild-type and thermostable mutants of kanamycin nucleotidyltransferase in Escherichia coli.
abstract: 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.
authors.affiliation: University of Wisconsin Biotechnology Center, Madison 53705.
authors.forename: H H
authors.initials: HH
authors.lastname: Liao
mesh_terms: D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D002479:Inclusion Bodies / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011487:Protein Conformation; D012995:Solubility; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase