Syngene’s comprehensive gene-to-protein services start with cloning the gene and extend up to the supply of ready-to-use proteins. Proteins can be supplied as per the specific needs of the client ranging from mg to gram scale and varying from research grade to highly pure crystallography grade.
A typical gene-to-protein program includes codon optimization and synthesis, screening of expression in different host systems (bacteria, yeast, insect cells, mammalian cells), expression optimization, expression scale up, purification and quality analysis.
Protein expression
Syngene has the capability to produce a wide range of proteins including immunogens for antibody campaigns, in vitro and in vivo studies, IHC, structural biology, and conjugated reagents for bioassays. Our expertise extends across various host systems including bacterial, yeast, insect and mammalian, and we have well worked out novel expression strategies for high titers.
Expression in bacteria
- Bacterial hosts such as E. coli, Bacillus
- Expression screening in E. coli to identify suitable host strain and optimal conditions for expression
- Scale of expression: 2 – 10 L lab scale and 20 – 225L pilot scale
Expression in yeast
- Hosts such as Saccharomyces sp. and Pichia pastoris
- Expression screening to identify suitable host strain and optimal conditions for expression
- Scale of expression: 2 – 10 L lab scale and 20 – 225 L pilot scale
Expression in insect cells
- Insect Cell Lines used: Sf9, Sf21, High Five
- Generation of high titer recombinant baculovirus
- MOI studies
- Scale of expression: 2 to 50L in Wave Bioreactor
Expression in mammalian cells
- Mammalian cell lines: CHO-K1, HEK 293, CHO-S, CHO-DG44 etc.
- Transient transfection for expression and purification of proteins
- Generation of stable cell lines expressing recombinant proteins
- Scale of expression: 2 – 10 L lab scale and 30 – 300 L pilot scale
Protein purification
Syngene uses high throughput and automated purification systems enhanced by the use of state-of-the-art chromatography systems (Akta) to deliver purification of mg to gram quantities of proteins for various applications (research, assay, animal studies, crystallography). Our scientists are adept at:
- Development and optimization of purification strategy using multiple steps of chromatographic methods:
- Affinity
- Hydrophobic
- Ion exchange
- Size exclusion
- Purification of recombinant proteins containing designed purification tags: His6, GST, MBP, SUMO, FLAG, etc.
- Option of tag removal by proteolytic digestion (Thrombin, TEV, PreScission, SUMO protease, Factor Xa, etc.)
- Purification from inclusion bodies:
- Development of refolding methods by exhaustive screening of refolding conditions
- Refolding scale up and purification of properly folded protein
Protein characterization, analysis and quality control
Expertise across multiple techniques that are used to analyze each batch of protein produced that ensures strict quality control and minimal batch to batch variation:
- Electrophoresis (SDS-PAGE, native-PAGE)
- Capillary gel electrophoresis (CGE)
- DOT/Western blot
- Imaging capillary electrophoresis (iCE)
- Mass spectrometry (ESI-MS/MS, MALDI-TOF/TOF)
- Peptide mass finder printing
- Reverse Phase-HPLC
- Size Exclusion HPLC
- Glycoform analysis
- Endotoxin level detection
- Biological activity assays
Protein crystallography
Syngene’s protein crystallography services aim at providing a one-stop-solution for gene-to-structure programs. In collaboration with the strong bioassay and medicinal chemistry groups of Syngene, the crystallography facility is designed to support any structure-based drug design program. The crystallography services offered are:
- Crystallization screening and optimization
-
- Commercially available screens
- Customized screens
- Sitting drop, hanging drop, micro batch (under oil) and seeding (mosquito crystallization robot)
- Microscopic examination of drops, staining, mass spectroscopy of harvested crystals (MALDI)
- Electrophoresis, preliminary X-ray diffraction
- Custom screening
- Crystallization of protein-inhibitor/ligand complexes (co-crystallization)
- Soaking of crystals in inhibitor/ligand solution
- Cryoprotectant screening by preliminary X-ray diffraction
- Complete X-ray data collection using rotating anode source
- Synchrotron data collection (mail-in format)
- Molecular replacement and structure refinement
- Structure analysis and data presentation
Infrastructure
Syngene has a sophisticated biology facility with high-end instrumentation. We have an experienced team with proven track record of supporting discovery and development programs under biology capabilities.
Some of the key instruments are listed below:
Protein purification
- ÄKTA- FPLCs (START,Primes, Purifiers & Explorers)
- High speed centrifuges
- Ultra-centrifuge
- Sonifier
- Micro-fluidizer
- Lyophilizer
- Tangential/ cross flow concentrators
- Columns & resins covering broad range of chromatographic separations
Protein crystallography
- Mosquito 5-deck crystallography robot
- Polarizing light microscope
- Cryo-storage
- Other crystallography tools
Analytical
- BIAcore T200
- Imaging capillary electrophoresis (ICE)
- Capillary zone electrophoresis (CZE)
- IEF
- ELISA reader (96 well format)
- Karl Fischer coulometric titrator
- Chemi Doc-XRS (Bio-Rad)
- RP-HPLC
- Image Quant 300; Image scanner
- Mass spectrometry (MALDI/TOF-TOF)
- ESI-MS with HMD
- Octet RED
- WES (Western blotting)
