Nanobodies and SUMO

Although mammalian cells have been proven as workhorses for antibody production, engineered single chain antibodies (nanobodies) are especially difficult to manufacture in mammalian cells. Many therapeutic programs are abandoned due to the lack of adequate and cost-effective expression. The engineered single chained antibodies express well as SUMO fusion in E.coli due to the chaperoning properties of SUMO to promote correct folding and protect the fused nanobody from degradation. These factors contribute to dramatically enhance protein production and improve the quality of nanobodies. The SUMO system is the most cost-effective way to generate the desired N-terminus of the protein. Nature designed SUMO to chaperone proteins in the cell. LifeSensors can guide scientists to the most suitable SUMO tag and SUMO protease to efficiently remove SUMO to reduce the cost of goods. LifeSensors can guide you from your cloning strategy to selection of strains/cell lines for the best expression and establish a non-GMP process from gene to nanobody. The process can be easily adapted for downstream GMP production of proteins.

The SUMO Tag

SUMO (Small Ubiquitin-like Modifier) is a member of the ubiquitin family, composed of a flexible N-terminal region followed by a ball-shaped ubiquitin-like fold. SUMO’s hydrophobic core improves correct folding of the fused nanobody, and its hydrophilic surface keeps the nanobody soluble. SUMO proteases recognize SUMO structure to cleave at the junction to generate the desired N-terminus of the nanobody.

SUMO Expression with Nanobodies

Figure 2: The Principle of the system. The 6xHis is used to assist in purification while SUMO significantly improves expression. SUMO Protease cleaves the SUMO 6xHis tag to separate the expressed Nanobody.

the protein structure of SUMO

Figure 1: The unique string and ball structure of SUMO allows for improved expression due to a hydrophilic outer surface and a hydrophobic core.

Many Nanobodies have already been expressed through SUMO systems:

  • Fibronectin type III domain based monobodies were expressed and purified using the SUMO fusion system in E.coli.[1]
  • Fab fragments: Cytosolic expression of functional fab fragments in E. coli was made possible using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode[2]
  • Single-chain variable fragment (scFv): A high level expression of an anti-VEGF165 scFv in E. coli was achieved through SUMO fusion.[3]
  • Nanobodies against GPCR, PAI, and SARS-CoV-2 RBD were expressed and purified in E. coli[4],[5],[6],[7],[8]
  • VHH antibodies: Single-Domain VHH antibodies against FMDV proteins and Chikungunya E2 protein were expressed and purified with the SUMO fusion system.[9][10][11]
  • Heavy-chain-only antibodies (HCABs): HCABs against influenza HA protein were produced in E.coli via a SUMO fusion.[12]
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[1] Gorman K et al, Curr Protoc Chem Biol. 2018; Hussain M et al, Biochemistry. 2018
[2] Rezaie F et al, J Appl Microbiol. 2017
[3] Sillen M et al, Int J Mol Sci. 2020
[4] Koehl A et al, Nature. 2019
[5] Sillen M et al, Int J Mol Sci. 2020
[6] Shi Z et al, Structure. 2022
[7] Sillen M et al, J Thromb Haemost. 2020
[8] Sillen M et al, Int J Mol Sci. 2021
[9] Li H et al, Vaccines (Basel). 2021;
[10] Li Q et al, J Nanobiotechnology. 2022
[11] Wang D et al, BMC Vet Res. 2015
[12] Drabek et al, Front Immunol. 2016

Lifesensors has pioneered SUMO technologies and discovery, and is looking forward to sharing these discoveries and applications with the greater pharmaceutical and biotech communities.

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