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Expression of Difficult Proteins

Many proteins are extremely difficult to express in heterologous expression systems. A vast number of factors may contribute to this problem. A common problem is that it can often be challenging for a foreign host to correctly fold a protein it does not normally produce. For example, in many experimental scenarios expression of a protein originating from a higher eukaryote is being produced in a bacterium where factors such as codon usage, translation rate, and redox potential are significantly different. Additionally, inherent properties of the target protein may represent challenges for the expression host. For example, a protein having multiple membrane spanning domains might not properly insert into membrane bilayers of the heterologous host or a protein might not be expressed in a soluble form. Finally, many proteins require post-translational modifications (e.g. glycosylation or phosphorylation) that are absent or significantly different from expression host to expression host.

There is no single solution for the expression of all classes of difficult proteins. Instead, expression problem-specific solutions that aim to better the chances of success can be used. For microbial expression systems, these solutions often come in the form of unique host strains that have been genetically modified to enhance the production of a certain difficult protein class. Other expression solutions seek to address problems by controlling aspects of how a target protein is produced. For example, some expression hosts allow for precise control of target gene expression. In addition, certain protein tags can help a protein to more efficiently insert into a host membrane or improve the solubility of a target protein.

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Expression of Difficult Proteins includes these areas of focus:
Disulfide-bonded Protein Expression
Membrane Protein Expression
Toxic Protein Expression
Target Protein Insolubility
FAQs for Expression of Difficult Proteins
Protocols for Expression of Difficult Proteins
Application Notes for Expression of Difficult Proteins
    Publications related to Expression of Difficult Proteins
  1. Agrawal, A., Bisharyan, Y., Papoyan, A, Bednenko, J., Cardarelli, J., Yao, M., Clark, T., Berkm​en, M., Ke, N., Colussi, P. 2019. Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli. Protein Expr Purif. 153, PubMedID: 30081196, DOI: 10.1016/j.pep.2018.08.001.
  2. Leith, E.M., O'Dell, W.B., Ke, N., McClung, C., Berkmen, M., Bergonzo, C., Brinson, R.G., Kelman, Z 2019. Characterization of the internal translation initiation region in monoclonal antibodies expressed in Escherichia coli J Biol Chem. 294(48), PubMedID: 31604819, DOI: 10.1074/jbc.RA119.011008
  3. Reddy, P.T., Brinson, R.G., Hoopes, J.T., McClung, C., Ke, N., Kashi, L. 2018. Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli. mAbs MAbs. 10 (7), PubMedID: 30060704, DOI: 10.1080/19420862.2018.1496879
  4. Ke, Na; Berkmen, Mehmet; Ren, Guoping; 2017. A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo Nat Chem Biol. 13, PubMedID: 28628094, DOI: 10.1038/nchembio.2409
  5. Ren, G., Ke, N. and Berkmen, M. 2016. Use of the Shuffle Strains in Production of Proteins. Curr Protoc Protein Sci. Aug 1, PubMedID: 27479507 , DOI: 10.1002/cpps.11.
  6. Anton, B.P., Fomenkov, A., Raleigh, E.A. and Berkmen, M. 2016. Complete Genome Sequence of the Engineered Escherichia coli SHuffle Strains and Their Wild-Type Parents Genome Announc. Mar 31;4(2), PubMedID: 27034504, DOI: 10.1128/genomeA.00230-16.
  7. Robinson, M.-P., Ke, N., Lobstein, J., Peterson, C., Szkodny, A., Mansell, T.J., Tuckey, C., Riggs, P.D., Colussi, P.A., Noren, C.J., Taron, C.H., Delisa, M.P., Berkmen, M. 2015. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria Nat Commun. (6)8072, PubMedID: , DOI: 10.1038/ncomms9072.
  8. Chatelle C, Kraemer S, Ren G, Chmura H, Marechal N, Boyd D, Roggemans C, Ke N, Riggs P, Bardwell J, Berkmen M 2015. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase Antioxid Redox Signal. , PubMedID: 26191605, DOI: 10.1089/ars.2014.6235
  9. Berkmen, M. 2012. Production of disulfide-bonded proteins in Escherichia coli Protein Expr Purif. , PubMedID: 22085722, DOI:
  10. Shouldice, S.R., Cho, S.H., Boyd, D., Heras, B., Eser, M., Beckwith, J., Riggs, P., Martin, J.L.and Berkmen, M. 2010. In vivo oxidative protein folding can be facilitated by oxidation-reduction cycling. Mol Microbiol. 75(1), PubMedID: 19968787, DOI:
  11. Manta, Bruno; Berkmen, Mehmet; . Disulfide Bond Formation in the Periplasm of Escherichia coli EcoSal Plus. , PubMedID: , DOI: 10.1128/ecosalplus.ESP-0012-2018.
Improvement of Protein Solubility with Lemo21(DE3)
A) B. malayi protein expressed at 20°C in BL21(DE3).
B) Soluble fractions of B. malayi protein expressed at 30°C in Lemo21 (DE3).
Disulfide Bond Formation
Disulfide bond formation in the cytoplasm of wild type E. coli is not favorable, while SHuffle is capable of correctly folding proteins with multiple disulfide bonds in the cytoplasm.
vtPA Expression in SHuffle®
Truncated tissue plasminogen activator (vtPA), which contains nine disulfide bonds when folded and oxidized correctly, was expressed from a pTrc99a plasmid in the cytoplasm of E. coli cells. After induction, cells were harvested and crude cell lysates were prepared. vtPA was assayed using a chromogenic substrate Chromozym t-PA (Roche #11093037001) and standardized to protein concentration using Bradford reagent. E. coli wt + cells are DHB4, which is the parent of FÅ113 (Origami™).
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    Solutions for the Expression of Difficult Proteins

    NEB has a long history in recombinant protein expression and has developed a wide array of solutions for proteins that are difficult to express.

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