Cloning and expression of the branching enzyme gene (glgB) from the cyanobacterium Synechococcus sp. PCC7942 in Escherichia coli.
Using the glgB gene from Escherichia coli as a hybridization probe, the gene encoding the branching enzyme of the cyanobacterium Synechococcus sp. PCC7942 has been identified on a 3.9-kb PstI fragment which was cloned into plasmid pUC9. Two types of plasmids have been isolated. Plasmid pKVN1 was expressing the Synechococcus sp. gene as was shown by complementation of the glgB mutation of E. coli KV832. Plasmid pKVN2, which carried the same insert in the opposite orientation was unable to complement E. coli KV832, indicating that the promoter of the cloned gene was either absent or was not recognized in E. coli. Determination of branching activity in extracts of Synechococcus sp. and E. coli KV832[pKVN1] showed that the enzyme was optimally active at approximately 35 degrees C. No significant activity was present at temperatures higher than 55 degrees C, reflecting the mesophilic nature of the cloned enzyme. In a cell-free coupled transcription-translation system the cloned gene specified two proteins of 84 kDa and 72 kDa, respectively, which are probably translated independently from the same gene by initiation at two different start codons.
Kiel
J A
JA
Department of Genetics, Center of Biological Sciences, Haren, The Netherlands.
Elgersma
H S
HS
Beldman
G
G
Vossen
J P
JP
Venema
G
G
D015061:1,4-alpha-Glucan Branching Enzyme / Q000235:genetics* / Q000378:metabolism; D015139:Blotting, Southern; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005964:Glucosyltransferases / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015183:Restriction Mapping; D013696:Temperature; D014158:Transcription, Genetic
D004247:DNA; D005964:Glucosyltransferases; D015061:1,4-alpha-Glucan Branching Enzyme