The solubility and stability of recombinant proteins are increased by their fusion to NusA.
The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13-20mg of fusion protein per litre of culture and 2.5-5mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins.
De Marco
Valeria
V
EMBL Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Stier
Gunter
G
Blandin
Stephanie
S
de Marco
Ario
A
D004355:Drug Stability; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D035602:Transcriptional Elongation Factors
D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D005982:Glutathione Transferase