enGenes X-press achieves high yield SARS CoV-2 nucleocapsid protein expression

Scientists from recombinant proteins specialist CDMO enGenes Biotech GmbH (enGenes) have collaborated with researchers from Austria and Belgium in a study that indicates enGenes X-press® technology has a vital part to play in isolation of the SARS-CoV-2 nucleocapsid protein that is central to the battle against the COVID-19 pandemic.

The study demonstrated how nucleocapsid protein could be produced in high titers using X-press as a growth-decoupled expression system, using CASPON tag technology to enable high cleavage specificity and developing a novel analytical HIC-MALS for superior quality control. The team also showed how  an optimized downstream processing (DSP) set-up could ensure 95% product purity.

Implications for vaccine campaign

All these advances are key to more effective COVID-19 testing as well as vaccine development. Using SARS CoV-2 in serological assays allows for distinguishing between COVID-19 antibodies produced by infection and those conferred by vaccination with SARS CoV-2 Spike-based vaccines.

enGenes CEO Dr. Juergen Mairhofer and co-founder Dr. Gerald Striedner joined fellow researchers representing four other Vienna-based institutions; the Department of Biotechnology of University of Natural Resources and Life Sciences, Vienna (BOKU) and its Mass Spectrometry Core Facility, the Austrian Centre of Industrial Biotechnology (acib), and bioprocess modeling specialists NovaSign GmbH; in the study, which was led by Dr. Jelle De Vos, from the Department of Chemical Engineering at Vrije University, Brussels and Dr. Nico Lingg, from the University of Natural Resources and Life Sciences, Vienna (BOKU).

The results of their study, ‘Production of full-length SARS-CoV-2 nucleocapsid protein from Escherichia coli optimized by native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection’ have been published in a recent issue of the renowned journal Talanta [1].

NP target

The nucleocapsid protein (NP) plays a critical role in the viral life cycle of SARS-CoV-2 and is abundantly expressed during infection, making it an ideal diagnostic target. NP has a strong tendency for dimerization and interaction with nucleic acids, making it problematic to express and purify.

This study succeeded for the first time in expressing high titers of NP in E. coli with a CASPON tag, using the growth-decoupled protein expression system, enGenes X-press. Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of DSP steps.

HIC-MALS in-process control

In-process control was established by using an analytical method consisting of native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS). This allowed monitoring of product fragmentation and multimerization throughout the purification process.

This optimised process succeeded in producing 730 mg purified NP per liter of fermentation, corresponding to a yield of 77% after cell lysis, with HIC-MALS indicating NP product purity of 95%, verified by peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay. The presence of a minor endogenous chaperone explained the residual impurities. A complementary native size-exclusion separation (SEC)-MALS analysis confirmed that molecular mass of the main NP fraction was consistent with dimerized protein.

The optimized HIC-MALS method not only enables monitoring of the product purity, with simultaneous access to its molecular mass bu also provides orthogonal information that complements established SEC-MALS methods, with superior resolving power over SEC,  attributed to the extended variables to tune selectivity in HIC mode.

Powerful evaluation tool

“The HIC-MALS method is a powerful analytical tool that was used for evaluation of the end product resulting from a biotechnological production platform that features growth-decoupled expression in the E. coli enGenes-X-press strain, and the CASPON platform process,” the team reports.

They suggest that the HIC-MALS-based method can form a powerful tool to guide process development of proteins produced from recombinant origin under native conditions.

“Extended information can be obtained by online coupling of a refractive index detector with MALS, provided that proper calibration and baseline subtraction is performed. With this native analytical method, the availability of high-quality antigens for further research and diagnostic purposes can be accelerated, especially in highly demanding times, such as the current COVID-19 pandemic,” the study concludes.


  1. De Vos, J., Patricia Pereira Aguilar, P., Köppl, C., Fischer, A., Grünwald-Gruber, C., Mairhofer, J., Striedner, G., Dürkop, M., Klausberger, M., Cserjan-Puschmann, M., Jungbauer, A., Lingg, N., (2021), Production of full-length SARS-CoV-2 nucleocapsid protein from Escherichia coli optimized by native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection’; Talanta, vol. 211, Article 120673. DOI: https://doi.org/10.1016/j.talanta.2021.122691

About enGenes Biotech

enGenes Biotech GmbH (enGenes) is a contract research, development and manufacturing company that provides leading edge technologies and production services focused on recombinant proteins in bacteria. The company’s mission is to provide cost-effective and scalable production of recombinant proteins at a fraction of current cost, allied to a vision of developing a world-class portfolio of cutting-edge protein production technologies, relevant to a broad spectrum of application fields.

enGenes has developed advanced technologies to drive more cost-effective recombinant protein production processes, including its proprietary enGenes-X-press™ E. coli platform that achieves outstanding yields of soluble and active recombinant protein. enGenes-X-press™ has been successfully applied for the manufacturing of enzymes and biopharmaceutical products that failed to give economically feasible yields in a conventional expression host.

enGenes Biotech offers development and manufacturing services tailored around the needs of pharmaceutical and industrial biotech companies. The services include expression strain and vector development, fermentation process development and optimization, downstream process development, production of purified protein, technology transfer and scale-up support with technology out-licensing and co-development opportunities.


Click on Production of full-length SARS-CoV-2 nucleocapsid protein from Escherichia coli optimized by native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection to view full study at Science Direct.

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