Recombinant proteins fused to thermostable partners can be purified by heat incubation.
We developed a protocol for the fast purification of small proteins and peptides using heat incubation as the first purification step. The proteins are expressed from a new bacterial expression vector (pETM-90) fused to the C-terminus of thermostable Ftr from Methanopyrus kandleri. The vector further contains a 6xHis-tag to allow immobilised metal ion affinity purification and a TEV protease cleavage site to enable the removal of the His-tag and fusion partner. Heat incubation induces the specific denaturation and precipitation of the Escherichia coli proteins but not of the thermostable fusion protein. Using the fusion construct and the heat incubation protocol a number of fusion proteins were purified to near homogeneity. The thermostability was ensured when Ftr had a molecular weight higher than twice the target protein. The obtained purification yields were similar and, in some cases, even higher than the ones obtained by affinity purification with the same Ftr-fusion proteins or the same target proteins fused to other often used partners such as NusA, GST, or DsbA. The protocol does not depend on a specific thermostable protein as was shown by the exchange of Ftr for M. kandleri Mtd. Purification by heat incubation is a fast and inexpensive alternative to chromatographic techniques, particularly suitable for the production of antigenic sequences for which the loss of native structure is not detrimental. We proved that it can be easily automated.
de Marco
Ario
A
European Molecular Biology Laboratory, Heidelberg, Germany. ademarco@embl-heidelberg.de
Casatta
Elisabetta
E
Savaresi
Sara
S
Geerlof
Arie
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019605:Euryarchaeota / Q000201:enzymology; D005240:Feasibility Studies; D005822:Genetic Vectors / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006358:Hot Temperature; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D006639:Histidine; D014166:Transferases; D010450:Endopeptidases; C099488:TEV protease