Disulfide bonds play structural, catalytic, and signaling roles in plants. Eukaryotic expression systems such as Chinese hamster ovary (CHO), yeast, or insects that can be used to produce disulfide-bonded proteins are slow and expensive. For most applications, prokaryotic expression systems remain a low-cost, fast, high-yield genetic tool that can be used for optimization purposes.

However, E. coli inhibits the formation of disulfide bonds in proteins due to the inherent reducing environment. Therefore, CD BioSciences provides customers with strategies for developing soluble expressions of disulfide-bonded proteins and peptides in bacteria. 

 Figure 1. Periplasmic disulfide bond formation process. (Berkmen M., 2012)

Service Content

Disulfide bond type analysis service

We provide our clients with protein sequence analysis services to determine whether the type of disulfide bond belongs to a structural, signaling, or catalytic bond. We can also

• Analyze the number of conserved cysteines.
• Detection of the extracytoplasmic location of proteins.

Periplasmic expression disulfide bond protein analysis service

• Regulation of expression levels.

Considering the potential danger of cytoplasmic overcrowding, we can efficiently regulate protein secretion at the translational level by modifying the Shine-Dalgarno sequence. In addition, our service includes the evaluation of parameters about the production rate of disulfide-bonded proteins, such as

• Effect of growth medium on production rate.
• Effect of plasmid replication start point on production rate.
• Effect of expression promoter on production rate.

Increased disulfide bond protein production is achieved by reducing protein precipitation and cell lysis through our services.

• Selecting the right precursor peptide for secretion.
• Increasing the rate of protein oxidation in the periplasm.
• Co-expression of periplasmic chaperones and folding enzymes.
• High-throughput selection of suitable clones.

Protein accumulation within the population varies by cell, so we utilize flow cytometry to select the most productive clones for our clients.

Cytoplasmic expression disulfide bond protein analysis service

• Screening carriers for improving protein folding accuracy.
• Oxidative refolding of denatured proteins.

Our services include screening

• Refolding buffer with redox pair and basic pH.
• The appropriate temperature for reducing sub-stable folding intermediates.
• The optimal concentration for refolding of heavy chain single domains.
• Detection of the redox state of disulfide isomerase (DsbC) in the cytoplasm.
• Evaluate the role of DsbC in the formation of polydisulfide bond proteins.
• Changing the redox state of cells.

We provide customers with the ability to alter the redox state of bacterial cytoplasm to allow it to undergo oxidative folding and form stable disulfide bonds within its cytoplasm.

• Optimization of protein expression conditions.

We can optimize the reaction conditions for customers, including

• Temperature
• Induction time
• Inducer concentration (IPTG)
• Solubility analysis of expressed disulfide bond proteins.

Disulfide bond protein purification service

We use Ni-NTA superfluid resin for rapid purification of soluble disulfide bond proteins from cell lysates. RP-HPLC column chromatography is used for final purification and to characterize the purity of target proteins.

Development service of Cell-Free Protein Synthesis (CFPS) platforms

We offer development services of CFPS platforms to facilitate the production of disulfide-bonded proteins in a rapid and economically viable format.

CD BioSciences is a biotechnology company focused on plant protein research, providing a variety of related services and products for environmental and energy solutions. If you are interested in our services, please contact us for more details. 

For research use only, not for clinical use.