Development of Thermus-Escherichia shuttle vectors and their use for expression of the Clostridium thermocellum celA gene in Thermus thermophilus.
We describe the self-selection of replication origins of undescribed cryptic plasmids from Thermus aquaticus Y-VII-51B (ATCC 25105) and a Thermus sp. strain (ATCC 27737) by random insertion of a thermostable kanamycin adenyltransferase cartridge. Once selected, these autonomous replication origins were cloned into the Escherichia coli vector pUC9 or pUC19. The bifunctional plasmids were analyzed for their sizes, relationships, and properties as shuttle vectors for Thermus-Escherichia cloning. Seven different vectors with diverse kanamycin resistance levels, stabilities, transformation efficiencies, and copy numbers were obtained. As a general rule, those from T. aquaticus (pLU1 to pLU4) were more stable than those from the Thermus sp. (pMY1 to pMY3). To probe their usefulness, we used one of the plasmids (pMY1) to clone in E. coli a modified form of the cellulase gene (celA) from Clostridium thermocellum in which the native signal peptide was replaced in vitro by that from the S-layer gene of T. thermophilus HB8. The hybrid product was expressed and exported by E. coli. When the gene was transferred by transformation into T. thermophilus, the cellulase protein was also expressed and secreted at 70 degrees C.
Lasa
I
I
Centro de Biología Molecular, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Spain.
de Grado
M
M
de Pedro
M A
MA
Berenguer
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D016963:Thermus thermophilus / Q000235:genetics*
D002480:Cellulase