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Cited by 20RNA and protein synthesis mechanismsProtein purification and stabilityMonoclonal and Polyclonal Antibodies Research

Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli

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Kulmala, Antti, Huovinen, Tuomas, Lamminmäki, Urpo·2017·Scientific Reports
DOI 10.1038/s41598-017-03957-6PMC PMC5476587
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Abstract

Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.

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  • At the transcriptional level, codon harmonization has largely improved expression of antibody fragments.

    “At the transcriptional level, the concept of “codon harmonization”, a sophisticated version of the codon usage optimization, has largely improved the expression of antibodies fragments [60,103].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [60]

    The Abstract and Discussion report that codon harmonization increased expression of a human Fab fragment in E. coli from negligible levels to 10 mg/l. The claim generalizes that result to antibody fragments and places the effect at the transcriptional level; the Discussion instead describes a suggested mechanism involving translation kinetics and protein folding. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • A recovery of 10 mg/mL of highly immunoreactive Fabs was obtained using pelB, the BL21 strain and the SB medium in combination with a harmonizing DNA approach.

    “In another study, a recovery of 10 mg/mL of highly immunoreactive Fabs was obtained by using pelB, the BL21 strain and the SB medium in combination with a harmonizing DNA approach [60].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [60]

    The Methods (Up-scaled protein production) describe harmonized Fab0 produced in BL21 using SB medium, and the Results (Characteristics of the parent genes and the genetic variants) identify a PelB signal sequence. But the Results (Up-scaled protein production) report 11.0 and 9.5 mg/L of functional Fab—not 10 mg/mL. The claimed concentration is 1,000 times higher than the approximately 10 mg/L reported. (LLM, conf=0.91)

    Experimental — accuracy not yet measured

  • Kulmala and coworkers demonstrated that harmonized versions of Fab fragments rather than the classic over-optimized one increase expression from negligible levels to 10 mg/L.

    “Recently, Kulmala and coworkers have demonstrated that the “harmonized versions” of Fab fragments rather than the classic “over-optimized” one increases the expression from negligible levels to 10 mg/L [60].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [60]

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Authors
Kulmala, A., Huovinen, T., & Lamminmäki, U.
Affiliations
  • University of Turku (FI)
  • University of Cambridge (GB)
Corresponding author
  • Kulmala, Antti
  • Lamminmäki, Urpo
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-06-19
Volume
7
Issue
1
Pages
3796-3796
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-017-03957-6
PMC
PMC5476587
OpenAlex
W2626530757
Cited by
20
References
4
Citations by year
2025: 1 · 2024: 2 · 2023: 2 · 2022: 3 · 2021: 4 · 2020: 4 · 2019: 3 · 2018: 1
FWCI
0.89
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Turun Yliopisto
  • Turun yliopiston tutkijakoulu
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
Keywords
antibody Fab fragment, codon optimization, recombinant protein expression, Escherichia coli toxicity, filamentous phage display, ampicillin resistance, protein yield
MeSH terms
Escherichia coli, Immunoglobulin Fab Fragments, Recombinant Proteins, Gene Expression

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