Post-translational modification(s) and cell distribution of Streptomyces aureofaciens translation elongation factor Tu overproduced in Escherichia coli.
We cloned EF-Tu from Streptomyces aureofaciens on a pET plasmid and overproduced it using the T7 RNA polymerase system in Escherichia coli. Streptomyces EF-Tu represented more than 40% of the total cell protein and was stored mostly in inclusion bodies formed apically at both ends of E. coli cells. Analysis of the inclusion bodies by transmission and scanning electron microscopy did not reveal any internal or surface ultrastructures. We developed the method for purification of S. aureofaciens EF-Tu from isolated inclusion bodies based on the ability of the protein to aggregate spontaneously. EF-Tu present in inclusion bodies was not active in GDP binding. Purified protein showed a similar charge heterogeneity as EF-Tu isolated from the mycelium of S. aureofaciens and all of the isoforms reacted with EF-Tu antibodies. All isoforms also reacted with monoclonal antibodies against O-phosphoserine and O-phosphothreonine.
Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czechia.
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LD
Nguyen
M
M
Holub
L
L
Kalachová
M
M
Weiserová
J
J
Kormanec
O
O
Benada
O
O
Kofronová
J
J
Weiser
D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006153:Guanosine Diphosphate / Q000378:metabolism; D002479:Inclusion Bodies / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D010444:Peptide Elongation Factor Tu / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D020033:Protein Isoforms / Q000276:immunology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013304:Streptomyces aureofaciens / Q000235:genetics*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D010444:Peptide Elongation Factor Tu