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Bacterial Protein Expression

  • Gene synthesis
  • Mutation
  • Cleavage site construction
  • Tag Cleavage
  • Refolding
  • Fermentation

THE SERVICE AMSBIO PROVIDE
SCHEMATIC REPRESENTATION OF THE SERVICE AMSBIO PROVIDE
PHASE 1 : Cloning of your gene of interest into a bacterial expression vector with His-tag or other selected tag (such as GST, His, or Thioredoxin):
  • Amplification/isolation of the gene of interest out of a customer-supplied vector and subcloning it into a bacterial expression vector.
  • Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.
  • Transformation of recombinant constructs into a high efficiency expression bacterial strain.
  • Mini-induction to over-express the target protein.
  • Test for the expression of the recombinant protein by SDS-PAGE and/or western blot (customer must provide appropriate antibodies)if desired.
  • Estimated time: 2-4 weeks.



PHASE 2 : Large-scale culture and purification:
  • One litre of bacterial culture will be induced with IPTG and harvested for protein purification.
  • Protein purification using Ni-NTA (for 6xHIS-tagged proteins) or glutathione (for GST-tagged proteins) beads at either native or denatured conditions.
  • Solubility not guaranteed.
  • Estimated time: 2 weeks.



Products:

5 μg construct, protein, documents (sequencing data,Western blot & SDS-PAGE result, design form).
If project stops after phase 1, products will include the above items minus protein.

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