Our protein expressing platform is focused on E. coli host system. We have designed a serial of E. coli expression vectors that allow us to screen various methods  and various parameters to meet our customer’s needs. We have following E. coli expression methods and we have multiple host cells and vectors for each expression method:

Cytoplasmic fusion protein expression

Periplasmic soluble expression

Inclusion body expression & refolding

Cytoplasm Fusion Protein Expression/Purification

For cytoplasmic protein expression in E. coli, the major issue is formation of inclusion body. To overcome this issue we have constructed six different vectors with different fusion partner proteins. So we can screen for the best fusion protein expression system. We also have a set of mutant E. coli strains that may increase the solubility of some expressed protein in E. coli cytoplasm.

We offer the choice of having 6XHIS tags at protein C-terminus. A client also have the option to retain or cleave the fusion protein, which contain a 6X HIS tag at N-terminal. After the cleavage the fusion protein, the delivered protein product can have all native sequence except one Ser or Gly reside at protein N-terminal.

We typically can purify the protein to >90% purity with one affinity step.  Higher affinity can be achieved with more purification steps. The purified product is often in a range of 1mg to 1g.

We also offer an optional service to characterize the protein product, including the confirmation of protein sequence, determination of protein N- and C-termini and mapping of disulfide bonds. 

1.    E. coli Periplasmic Protein Expression & Purification

For proteins containing disulfide bonds, expressing in E. coli periplasm sometime may gives a higher yield of soluble, correctly folded proteins. However, it is difficult to predict which secretion peptide will give a high yield periplasmic expression. So we have constructed a serial of vectors with compatible target gene insertion sites to screen for an optimized periplasmic expression system.

The protein product delivered often have purity > 90%. The amount is often in the range of 1mg. to 1g.

We also offer optional characterization service for the delivered products such as determination of protein N-terminal to confirm the removal of signal peptide, and disulfide bond mapping to confirm the correct S-S linkage.

2.    Inclusion Body Expression, Refolding and Purification.

For some proteins that are difficult to achieve a high yield expression in E. coli cytoplasm or periplasm,  expression as inclusion body and refolding  might be the method of choice to a high yield expression in E. coli. We have a vector system that often gives a high yield inclusion body expression. The expressed protein can be of nature sequence except the N-terminal Met may not remove. The major difficulty in refolding is that a number of conditions need to be tested experimentally in order to find a working procedure.We’ll typically test 6 default refolding procedures that have a good chance to give folded soluble proteins. Then we’ll further optimize several parameters to increase the yield.

We offer optional service for characterize the products, including quantitative mapping of disulfide bonds and confirmation of the protein N- and C-termini.

3.    Optional Characterization service for expressed and purified protein products

We also offer an optional service to characterize the protein product, including the confirmation of protein sequence, determination of protein N- and C-termini and mapping of disulfide bonds.