Construction of an expression and site-directed mutagenesis system of haloalkane dehalogenase in Escherichia coli.
Haloalkane dehalogenase from Xanthobacter autotrophicus was efficiently expressed in Escherichia coli BL21 (DE3) and E. coli JM101. After introduction of restriction sites by PCR the haloalkane dehalogenase gene (dhlA) was translationally fused behind the T7 (phi 10), trc, and tac promoters. This resulted in expression at 30 degrees C up to 38 and 18% of the total soluble cellular protein with the T7 and trc promoters, respectively. Dehalogenase expression under control of the tac promoter was below 1% of the soluble cell protein, however. Aggregation of haloalkane dehalogenase into inclusion bodies was found during growth at 37 degrees C but not at 30 degrees C. Aggregates were also formed from intact enzyme upon incubation at 37 degrees C of cells or crude extracts containing active mature dehalogenase. The high level of expression resulted in a short purification procedure in which 30-35 mg highly enriched haloalkane dehalogenase was obtained from an 0.51 culture. For the production of single-stranded DNA an f1(+) origin was introduced in the T7 expression system.
Schanstra
J P
JP
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, The Netherlands.
Rink
R
R
Pries
F
F
Janssen
D B
DB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D015870:Gene Expression; D005822:Genetic Vectors; D006867:Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D006867:Hydrolases; C046137:haloalkane dehalogenase