Service packages:

  1. Cloning and expression of recombinant proteins in coli
  2. Protein production in coli and purification
  3. Crystallization screening of (recombinant) protein
  4. Crystal structure determination of (recombinant) proteins and protein/ligand complexes

1 and 3 are phase-1 services (necessary for specifying and starting phase-2 services 2 and 4). The services as defined are valid for 2017 & 2018. Specific wishes or changes in a specific service package can be negotiated on a case-by-case basis.

 

Cloning and expression of recombinant proteins in coli

 Standardly included:

  • Synthetic gene design for optimal cloning, or primer design when gene is provided
  • Cloning in in-house optimized expression vectors
  • 3 constructs per protein: tag-less, His-tag, His-SUMO-tag
  • DNA sequencing to verify constructs
  • Small scale expression tests at varying conditions (at 17°C and 30°C, in LB and TB media), analysis via Coomassie-stained SDS-PAGE (total expression, soluble and insoluble fraction)
  • Full report of results

Duration:      3-5 weeks

Extra possibilities (on demand):

  • Creating other expression constructs (other expression vectors, other fusion partners, secreted expression)
  • Use of other coli strains
  • Expression in other hosts (yeast, Bacillus)
  • Developing expression for several proteins in parallel

Protein production in E. coli and purification

Included:

  • Pilot expression and purification (one construct) at 50 ml culture scale for protocol development
  • Expression at 1 liter culture scale, aiming at a final yield of 10 mg pure protein
  • One or two step purification protocol, including affinity chromatography
  • Analysis of purification results by Coomassie-stained SDS-PAGE
  • Full report of results

 Duration:      2-4 weeks

 Extra possibilities (on demand):

  • Upscale production and purification to 1 g pure protein
  • Purification of other/multiple constructs (e.g. different tags)
  • Quality analysis of purified protein by dynamic light scattering, silver-stained SDS-PAGE, thermofluor assay, mass spectrometry, etc.
  • Assay development, kinetic characterization of purified protein, ligand binding measurements
  • Removal of tag to produce native, tag-less protein

Crystallization screening of (recombinant) proteins

 Standardly included:

  • Quality analysis of purified protein by dynamic light scattering and silver-stained PAGE (SDS, native)
  • If necessary: one additional purification step to improve protein quality (e.g. gel filtration)
  • Screening for initial crystallization conditions in 96-well plates using four commercially screens at two different temperatures and varying protein concentration
  • Imaging and evaluation of plates during ~2 months
  • Full report of results

Duration:      3-4 months

Specifications:

  • Minimal requirement: 2-5 mg pure protein (in protein storage buffer)

Extra possibilities (on demand):

  • More extensive pre-crystallization analysis: e.g. thermofluor assay, mass spectrometry
  • Additional crystallization screening of protein variants (different tag, no tag) and/or co-crystallization with ligands
  • Screening with additional commercial or custom-made screens

 

Protein crystal structure determination

 Standardly included:

  • Optimization of initial hits for growth of X-ray diffracting crystals
  • Cryo-preservation of crystals and cryo-storage
  • In-house X-ray diffraction analysis and data collection
  • Data processing, phasing, model building and refinement
  • Validation of the final structure
  • Full report of results, including coordinates and diffraction data of final crystal structure

Duration:      4 – 12 months (go/no go decision after 4 months)

Extra possibilities (on demand):

  • X-ray data collection at the synchrotron
  • Crystallographic binding studies with ligands, inhibitors, etc
  • Structure analysis

Specifications:

  • Initial crystallization conditions have to be available (e.g. from literature or service package 3)
  • Duration and costs will be variable depending on full specification of the project and whether structure determination requires straightforward or non-straightforward procedures.
  • Milestones will have to be specified before start of project
  • Go/no go decision after 4 months (in case no well diffracting crystals are obtained)

 

Available equipment and facilities

 Fully equipped molecular biology and biochemistry laboratory

  • Akta protein chromatography systems
  • Mosquito® nanoliter high throughput pipetting-crystallization robot.
  • Dynamic light scattering equipment
  • Crystallization drop imager
  • MicroSTAR rotating anode X-ray generator (Bruker) with image plate detector
  • High-end computers for crystallographic computing and structure analysis