The 3-phosphoglycerate kinase of the hyperthermophilic archaeum Pyrococcus woesei produced in Escherichia coli: loss of heat resistance due to internal translation initiation and its restoration by site-directed mutagenesis.
Heterologous expression of the gene coding for 3-phosphoglycerate kinase (PGK) of the hyperthermophilic archaeum, Pyrococcus woesei (Pw), in Escherichia coli (Ec) yielded only low recovery of recombinant PGK (re-PGK) in heat-precipitated crude extracts. Moreover, we noticed contamination with a 28-kDa protein, from which PGK could hardly be separated, even under stringent conditions after tagging the re-PGK with a His6-tag. The preparations contaminated with the 28-kDa protein showed an unexpectedly low thermal stability. Under the same conditions (85 degrees C, 30 min), however, the enzyme from the original organism was completely resistant to heat inactivation. As shown by size-exclusion chromatography, re-PGK forms tight associations with the 28-kDa protein, which was found to represent a C-terminal fragment of PGK and to arise as a product of internal translation initiation within the pgk gene. Mutations changing the internal ribosome-binding site effectively suppressed the production of the 28-kDa protein and restored the thermal stability of the Pw re-PGK.
Hess
D
D
Universität GH Essen, Germany.
Hensel
R
R
D001105:Archaea / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010442:Peptide Chain Initiation, Translational; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D010735:Phosphoglycerate Kinase