Screening for soluble expression of recombinant proteins in a 96-well format.
For future structural and functional genomics programs new tools will be required. The implementation of high-throughput (HTP) methods for protein production will be an essential element. Present HTP protein production developments in structural genomics are aimed at obtaining well-expressing and highly soluble proteins, which are preferred candidates for structure-function studies. Here, we describe a cheap and efficient procedure to identify well-expressing soluble proteins in Escherichia coli in a compact 96-well format. Reproducible lysis on filter plates, followed by a filtration step on 96-well filter plates, allows the efficient separation of inclusion bodies from the soluble fraction. In the following step a dot blot procedure using anti-RGS-His(4) antibody (Qiagen) to detect expression of recombinant His-tagged protein is applied allowing direct detection of the target protein in the soluble fraction. The method is well suited for automation and should be applicable to expression screening of most proteins and fusion domains to which specific antibodies are available.
Knaust
R K
RK
Department for Biochemistry and Biophysics, Stockholm University, Svante-Arrhenius-Väg 12, SE-106 91 Stockholm, Sweden.
Nordlund
P
P
D001709:Biotechnology / Q000295:instrumentation* / Q000379:methods*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005374:Filtration; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D016133:Polymerase Chain Reaction; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D036224:Receptor, EphB4; D036081:Receptors, Eph Family; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D015203:Reproducibility of Results; D012995:Solubility
D011994:Recombinant Proteins; D020794:Receptor Protein-Tyrosine Kinases; D036224:Receptor, EphB4; D036081:Receptors, Eph Family