Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB

Manufacture of recombinant proteins with functional tags

Many questions in modern biotechnology are to be answered by experiments involving the use of recombinant cloned transcripts. Hereby, the supply of specific proteins with custom-made linker or labeling functions, so-called functional tags, is getting more and more important. This is valid in particular for the interdisciplinary research field of interfacial- and nanotechnology and molecular biology, in which such molecular tools are used.

Custom-made proteins for applications in proteomics

Production of fusion proteins with functional tags: EGFP in E. coli.
Fig. 1: Production of fusion proteins with functional tags: EGFP (enhanced green fluorescent protein) in E. coli. Left: EGFP construct in E. coli pellets induced via IPTG. Right: control, non-induced EGFP construct.

By the methods of genetic engineering it is possible to insert almost any well-known gene from any organism in a suitable expression system so that the protein can be manufactured and purified. The Fraunhofer IGB offers the design and the production of such custom-made, biologically active proteins in the milligram scale as a research service. We are happy to provide you an offer according to your specifications (modification, expression system, degree of purity, scale, intended purpose).

Possible applications of fusion proteins:

  • Protein/protein interactions: Identification of interacting proteins (co-immunoprecipitation, two hybrid assays)
  • Representation of protein microarrays
  • Production of antibodies against native proteins
  • Use of labeled proteins (fusion proteins) for protein localization in mammalian cells / overexpression
  • Use of recombinant peptides and proteins in the clinical therapy

Cloning and expression strategy

SDS PAGE for controlling the protein purification process.
Fig. 2: SDS PAGE for controlling the protein purification process. Lane 1: Protein marker (size), Lane 2: Total protein after cell disruption (French Press), SDS-PAGE and Coomassie staining. Lane 3: Purified target proteins (EGFP-Intein-HIS and Intein-HIS).
  • Strategy planning
  • Data base search
  • Design of construct (fusion protein, selection of a suitable tag)
  • Selection of a suitable expression system (eucaryotic, procaryotic)
  • Specific primer design (loops, hairpins)
  • PCR amplification of the gene of interest
  • Enzymatic manipulations
  • Ligation / transformation into the appropriate cloning system
  • Screening for mutants
  • Evaluation of cloning success by sequencing
  • Expression of the target protein
  • Induction optimization
  • Cell disruption
  • Different purification steps
  • Renaturation
  • Determination of activity
  • Immobilization
  • Measurements via MALDI TOF MS

References

For example we have constructed the following fusion proteins and provided them as native proteins in the milligram scale:

  • The cytokine macrophage migration inhibitory factor (MIF) with N-terminal streptactin tag (MIF-StrepTag)
  • Enhanced green fluorescent protein / Intein /Chitin binding protein fusion protein (EGFP-Intein-CBD)
  • Enhanced green fluorescent protein / Intein /10 x Histidine-tag fusion protein (EGFP-Intein-HIS)