Generation and diversification of recombinant monoclonal antibodies

  1. Keith F DeLuca
  2. Jeanne E Mick
  3. Amy Hodges Ide
  4. Wanessa C Lima
  5. Lori Sherman
  6. Kristin L Schaller
  7. Steven M Anderson
  8. Ning Zhao
  9. Timothy J Stasevich
  10. Dileep Varma
  11. Jakob Nilsson
  12. Jennifer G DeLuca  Is a corresponding author
  1. Colorado State University, United States
  2. University of Geneva, Switzerland
  3. University of Colorado Anschutz Medical Campus, United States
  4. Northwestern University, United States
  5. University of Copenhagen, Denmark

Abstract

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

Data availability

All data generated during this study are included in the manuscript. We will also deposit the plasmid text files and maps on our institutional repository and AddGene.

Article and author information

Author details

  1. Keith F DeLuca

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  2. Jeanne E Mick

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  3. Amy Hodges Ide

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  4. Wanessa C Lima

    Geneva Antibody Facility, Faculty of Medicine, University of Geneva, Geneva, Switzerland
    Competing interests
    No competing interests declared.
  5. Lori Sherman

    CU Cancer Center Cell Technologies Shared Resource, University of Colorado Anschutz Medical Campus, Aurora, United States
    Competing interests
    No competing interests declared.
  6. Kristin L Schaller

    Department of Pediatric Hematology, Oncology and Bone Marrow Transplant, University of Colorado Anschutz Medical Campus, Aurora, United States
    Competing interests
    No competing interests declared.
  7. Steven M Anderson

    Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, United States
    Competing interests
    No competing interests declared.
  8. Ning Zhao

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7092-6229
  9. Timothy J Stasevich

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  10. Dileep Varma

    Department of Cell and Developmental Biology, Northwestern University, Chicago, United States
    Competing interests
    No competing interests declared.
  11. Jakob Nilsson

    The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4100-1125
  12. Jennifer G DeLuca

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    For correspondence
    jdeluca@colostate.edu
    Competing interests
    Jennifer G DeLuca, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3598-1721

Funding

National Institute of General Medical Sciences (R35GM130365)

  • Jennifer G DeLuca

National Institute of General Medical Sciences (MIRA R35GM119728)

  • Timothy J Stasevich

National Institute of General Medical Sciences (K99GM141453)

  • Ning Zhao

National Institute of General Medical Sciences (R01GM135391)

  • Dileep Varma

National Science Foundation (MCB-1845761)

  • Timothy J Stasevich

National Cancer Institute (P30CA046934)

  • Lori Sherman
  • Steven M Anderson

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, DeLuca et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,273
    views
  • 1,013
    downloads
  • 7
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Keith F DeLuca
  2. Jeanne E Mick
  3. Amy Hodges Ide
  4. Wanessa C Lima
  5. Lori Sherman
  6. Kristin L Schaller
  7. Steven M Anderson
  8. Ning Zhao
  9. Timothy J Stasevich
  10. Dileep Varma
  11. Jakob Nilsson
  12. Jennifer G DeLuca
(2021)
Generation and diversification of recombinant monoclonal antibodies
eLife 10:e72093.
https://doi.org/10.7554/eLife.72093

Share this article

https://doi.org/10.7554/eLife.72093

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Katherine A Senn, Karli A Lipinski ... Aaron A Hoskins
    Research Article

    Pre-mRNA splicing is catalyzed in two steps: 5ʹ splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (first and second step factors). We recently identified Fyv6 (FAM192A in humans) as a second step factor in Saccharomyces cerevisiae; however, we did not determine how widespread Fyv6’s impact is on the transcriptome. To answer this question, we have used RNA sequencing (RNA-seq) to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3ʹ SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only second step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the first step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3ʹ SS.

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Eyal Paz, Sahil Jain ... Abdussalam Azem
    Research Article

    TIMM50, an essential TIM23 complex subunit, is suggested to facilitate the import of ~60% of the mitochondrial proteome. In this study, we characterized a TIMM50 disease-causing mutation in human fibroblasts and noted significant decreases in TIM23 core protein levels (TIMM50, TIMM17A/B, and TIMM23). Strikingly, TIMM50 deficiency had no impact on the steady-state levels of most of its putative substrates, suggesting that even low levels of a functional TIM23 complex are sufficient to maintain the majority of TIM23 complex-dependent mitochondrial proteome. As TIMM50 mutations have been linked to severe neurological phenotypes, we aimed to characterize TIMM50 defects in manipulated mammalian neurons. TIMM50 knockdown in mouse neurons had a minor effect on the steady state level of most of the mitochondrial proteome, supporting the results observed in patient fibroblasts. Amongst the few affected TIM23 substrates, a decrease in the steady state level of components of the intricate oxidative phosphorylation and mitochondrial ribosome complexes was evident. This led to declined respiration rates in fibroblasts and neurons, reduced cellular ATP levels, and defective mitochondrial trafficking in neuronal processes, possibly contributing to the developmental defects observed in patients with TIMM50 disease. Finally, increased electrical activity was observed in TIMM50 deficient mice neuronal cells, which correlated with reduced levels of KCNJ10 and KCNA2 plasma membrane potassium channels, likely underlying the patients’ epileptic phenotype.