Protein Refolding Services
High level of protein expression in E. coli is often accompanied by the accumulation of the target recombinant proteins in insoluble aggregates called Inclusion Bodies (IB). While the exact mechanism of IB formation is not fully understood and may vary with different proteins and expression conditions, it is generally thought that target proteins are being made at such a rapid pace that the cellular translational machinery of E.coli is unable to assist in folding of the protein in its native 3D conformation, hence resulting in misfolding and ultimately, IB formation. These insoluble aggregates are non-functional and create hurdles in the path of researchers. Recovery of the biological activity of the target protein by an in vitro refolding process is a difficult task and is one of the most significant challenges facing a protein biochemist. GenScript's protein refolding services provide a solution to this problem.
Our confidence is backed by our proprietary refolding technology, which includes high-hydrostatic-pressure techniques, small-molecule additives, and column-refolding methods.
Protein refolding services are provided along with our bacterial protein service packages upon request. Over 95% of the inclusion bodies can be solublized and refolded. We provide refolded protein at purity of 85% or greater. The final output is dependent on the total amount of target protein comprising the aggregates. The refolded protein is delivered in customized buffer. The major elements of our protein refolding services include the following procedures:
- Evaluation of target proteins' biochemical and biophysical properties
- Refolding optimizations in the 1L system
- The particular refolding strategy is selected based on protein's sequence and the structural properties. Typically, our strategies include the following:
- Direct dilution
- Dialysis and diafiltration
- Small molecular assistants:
- Amino acids
- Polymers: micellar and liposomal
- Reducing/oxidizing reagents
- Salts: Na/K/Mg-Cl and (NH4)2SO4
- Saccharides: glucose, sucrose, and trehalose
- Artificial chaperones: detergent and non-detergent
- Matrix-assisted methods: SEC (Size Exclusion Chromatography), HIC (Hydrophobic Interaction Chromatography), affinity column and IEC (Ion Exchange Chromatography)
- Chaperone-mediated methods: ATP-dependent and ATP-independent
- Gradient methods: pH, temperature, hydrostatic pressure, and denaturant
1. A Protein denaturation and renaturation from inclusion bodies:
Fig. A-1 (left figure): Inclusion body preparation and denaturation
Lane 1: Total protein after sonication
Lane 2: The supernatant after centrifugation
Lane 3: Washed with 1M urea
Lane 4: Dissolved by 8M urea
Fig. A-2 (right figure): Refolded protein
2. B Fusion protein denaturation and renaturation from inclusion bodies:
Lane 1: Refolded tag-free A protein
Fig. B-1 (left figure): Inclusion body preparation and denaturation
Lane 1: The supernatant after centrifugation
Lane 2: The precipitation after centrifugation
Lane 3: Inclusion bodies dissolved by urea
Lane 4: Flow through
Lane 5: Washed by imidazole containing urea
Lane 6: Eluted by imidazole containing urea
Fig. B-2 (right figure): Refolded protein
Lane 1 and 2: Refolded B fusion protein
- OptimumGene™ Codon Optimization Technology: Our proprietary OptimumGene™ codon optimization technology comprehensively optimizes factors critical to transcription, translation, and co-translational protein folding to deliver the highest possible levels of expression in any system.
Minimum 10-fold increase in protein expression level
- Bacterial Expression: GenScript has developed BacPower™ technologies to tackle hard-to-express and hard-to-dissolve proteins. Guaranteed package (SC1253): 3 mg purified protein.
- Yeast Expression: Our yeast expression system employs stable and durable production strains that resemble mammalian protein processing, incorporating features of high yield, high productivity, and dramatic reductions in costs and time requirements.
- Insect Cell/Baculovirus Expression: GenScript has pioneered methods for the secretion of recombinant proteins from baculovirus-infected insect cells. Our services are suitable for academic institutions and both the pharmaceutical and biotechnology industries.
- Mammalian Cell Expression: Our proprietary 293 and CHO suspension system platform promotes fast production and delivery of recombinant proteins and monoclonal antibodies up to grams level.
- Stable cell line development: Custom stable cell lines for assay development, recombinant protein and monoclonal antibody production. We also provide comprehensive assay-ready cell line productions to preserve your time for higher value projects.
GenScript provides fast, professional protein synthesis services at very reasonable prices. By making it cost-effective to outsource protein production, GenScript has made it possible for my lab to focus on our own area of expertise and get more research done. The detailed planning, updates, and reports that GenScript provides all of the quality control that one could ask for. I strongly recommend GenScript's protein production service.
— Dr. Barry Bradford, Kansas State University, USA
GenScript can provide soluble refolded protein with at least 85% purity from 1L expression system, based on total target protein comprising the aggregates. The refolded protein is delivered in customized buffer with the protein purity and identity data determined by Coomassie-stained gels and western blot analyses using Tag or the customer's designated protein-specific antibody (must be provided by the customer).
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