Single molecule assays reveal differences between in vitro and in vivo synthesized beta-galactosidase.
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 beta-galactosidase. In addition the enzyme was synthesized in vitro with a C-terminal His(6) tag. The enzyme expression was also induced in these strains using isopropyl-beta-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 His(6) 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 His(6) 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 His(6) 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 His(6) tag alters the activity of beta-galactosidase.
Nichols
Ellert R
ER
Chemistry Department, University of Winnipeg, Winnipeg, MB, Canada R3B 2E9.
Craig
Douglas B
DB