Recombinant protein expression in e.coli|FAQ|zoonbio

should I use the E.coli expression system?

E.coli expression is by far the most cost-efficient and consistent production method. We have the capabilities to scale up from milligrams to grams. Although E.coli do not incorporate post-translational modifications into the recombinant protein, many E.coli produced proteins do show activity of the native protein (our robust catalogue of active recombinant proteins is evidence of this). However bacterial cells produce endotoxins, so if you plan to use the protein in any animal cells or assays, endotoxin removal is recommended.

What are the Advantages of e.coli system?

1.Simple,well-understood genetics.
2.Its very easy to genetically manipulate.
3.Culturing cost is minimal.
4.Expression is fast,since the doubling time is only 20-30 mins.
5.Well established labeling protocols for stability studies.
6.Established regulatory track record.
7.Fermentation, easy to scale up.
8.Inclusion bodies may be easy to purify.

What are the disadvantages of e.coli system?

1.Eukaryotic co- and posttranslationalmodifications not possible.
2.Codon usage is different from eukaryotes.
3.Producing insoluble proteins.

What are the technical difficulties of e.coli system?

1.How to realize the expression of a large number of soluble proteins?
2.How to realize glycosylation modification of protein?

How much experience does zoonbio have for custom protein services in e.coli?

We started providing custom protein service since 2011. Till the end of 2019, more than 2,100 batches of custom proteins were successfully produced and delivered.

What starting materials do I have to provide ?

All we need to start with is the sequence information of your target protein/gene. We can start with gene synthesis with codon optimized for protein expression, and deliver you the final protein product. But if you would like us to start with your plasmid/virus, you are welcome to send them to us.

How much protein can your team usually obtain from 1L pilot project in e.coli expression system?

In bacteria system, the average is 1-5 mg/L. Our highest record is 200 mg/L.

What if the protein can't be expressed and purified in E. coli system?

There are a few things zoonbio scientists will do to solve the problem:
(1) Codon optimization is recommended if it has not been;
(2) remove the transmembrane region and signal peptide region;
(3) try to express truncated domains of the protein;
(4) we can also try different tags to increase the expression level;
(5) we may recommend customer to try different expression systems (e.g. insect cell system for certain proteins).

What tags does zoonbio recommend for protein expression and purification?

The common tags to use are GST, MBP. Trx, TF, MBP, Nus, SUMO, Flag, Fc, and so on. Zoonbio scientists may recommend different tags for different proteins based on the protein's property and the customer's specific requirements. His tag, either alone or fused with other tags (e.g. His-GST, etc.), is the most common one used for purification.