Single Molecule Assays Reveal Differences Between In Vitro and In Vivo Synthesized β-Galactosidase

Abstract

Genomic DNA was extracted from wild-type Escherichia coli strains ATCC 35321 and 8677. The lac Z gene was amplified and used as a template for in vitro synthesis of β-galactosidase. In addition the enzyme was synthesized in vitro with a C-terminal His₆ tag. The enzyme expression was also induced in these strains using isopropyl-β-D-galactoside. Single enzyme molecule assays were performed using a capillary electrophoresis-based protocol on both the in vitro and in vivo synthesized enzyme. In vivo produced enzyme from strains 35321 and 8677 showed average combined turnover numbers for the 4 active sites of the individual enzyme molecules of 53,400 ± 18,400 (N = 139) and 34,300 ± 17,800 min⁻¹ (N = 181) respectively. Average combined turnover numbers of 35,800 ± 20,900 (N = 302) and 31,700 ± 17,700 min⁻¹ (N = 315) were obtained respectively for the in vitro synthesized enzyme from strain 35321 with the absence and presence of a C-terminal His₆ tag. For strain 8677, the average combined turnover numbers were 29,000 ± 17,900 (N = 288) and 25,200 ± 12,600 min⁻¹ (N = 240) respectively for the absence and presence of a C-terminal His₆ tag. The average combined turnover numbers of the enzyme from both strains synthesized in vivo and in vitro and with the presence and absence of a His₆ tag were found to differ significantly. This indicates that the in vivo and in vitro produced enzymes are not identical and the presence of a C-terminal His₆ tag alters the activity of β-galactosidase.