Skip to main content
Kent Academic Repository

Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study.

Arauzo‐Aguilera, Klaudia, Buscajoni, Luisa, Koch, Karin, Thompson, Gary, Robinson, Colin, Berkemeyer, Matthias (2023) Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study. Microbial Cell Factories, 22 (1). Article Number 104. E-ISSN 1475-2859. (doi:10.1186/s12934-023-02111-4) (KAR id:101507)


In the biopharmaceutical industry, Escherichia coli is one of the preferred expression hosts for large-scale production of therapeutic proteins. Although increasing the product yield is important, product quality is a major factor in this industry because greatest productivity does not always correspond with the highest quality of the produced protein. While some post-translational modifications, such as disulphide bonds, are required to achieve the biologically active conformation, others may have a negative impact on the product's activity, effectiveness, and/or safety. Therefore, they are classified as product associated impurities, and they represent a crucial quality parameter for regulatory authorities. In this study, fermentation conditions of two widely employed industrial E. coli strains, BL21 and W3110 are compared for recombinant protein production of a single-chain variable fragment (scFv) in an industrial setting. We found that the BL21 strain produces more soluble scFv than the W3110 strain, even though W3110 produces more recombinant protein in total. A quality assessment on the scFv recovered from the supernatant was then performed. Unexpectedly, even when our scFv is correctly disulphide bonded and cleaved from its signal peptide in both strains, the protein shows charge heterogeneity with up to seven distinguishable variants on cation exchange chromatography. Biophysical characterization confirmed the presence of altered conformations of the two main charged variants. The findings indicated that BL21 is more productive for this specific scFv than W3110. When assessing product quality, a distinctive profile of the protein was found which was independent of the E. coli strain. This suggests that alterations are present in the recovered product although the exact nature of them could not be determined. This similarity between the two strains' generated products also serves as a sign of their interchangeability. This study encourages the development of innovative, fast, and inexpensive techniques for the detection of heterogeneity while also provoking a debate about whether intact mass spectrometry-based analysis of the protein of interest is sufficient to detect heterogeneity in a product. [Abstract copyright: © 2023. The Author(s).]

Item Type: Article
DOI/Identification number: 10.1186/s12934-023-02111-4
Uncontrolled keywords: Fermentation, Single-Chain Antibodies - genetics - metabolism, Escherichia coli BL21 and W3110, Recombinant Proteins, Disulfides - metabolism, Product heterogeneity, Escherichia coli Proteins - metabolism, Escherichia coli - metabolism, Disulphide bond, Protein purification, Sec pathway
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: European Union (
SWORD Depositor: JISC Publications Router
Depositing User: JISC Publications Router
Date Deposited: 08 Jun 2023 10:28 UTC
Last Modified: 09 Jun 2023 08:45 UTC
Resource URI: (The current URI for this page, for reference purposes)

University of Kent Author Information

Arauzo‐Aguilera, Klaudia.

Creator's ORCID:
CReDIT Contributor Roles:

Robinson, Colin.

Creator's ORCID:
CReDIT Contributor Roles:
  • Depositors only (login required):

Total unique views for this document in KAR since July 2020. For more details click on the image.