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SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restricted Ablation In vitro) for Antibody Affinity Maturation and Paratope Mapping
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.ORCID-id: 0000-0002-7875-2822
KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.ORCID-id: 0000-0002-4858-8056
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.ORCID-id: 0000-0002-9977-5724
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Abstract [en]

Mutagenesis libraries are essential for combinatorial protein engineering. Despite improve- ments in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of intact antibody scFv genes and simultaneous diversification of all six CDRs. Here, we de- scribe the generation of mutagenesis libraries for antibody affinity maturation, using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. This procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, and elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with di- versity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed >99% functional diversity in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed quicker enrichment of improved binders compared to the other two diversification strategies.

Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
URN: urn:nbn:se:kth:diva-227189OAI: oai:DiVA.org:kth-227189DiVA, id: diva2:1203711
Anmärkning

QC 20180525

Tillgänglig från: 2018-05-04 Skapad: 2018-05-04 Senast uppdaterad: 2020-01-10Bibliografiskt granskad
Ingår i avhandling
1. Methods for cell line and protein engineering
Öppna denna publikation i ny flik eller fönster >>Methods for cell line and protein engineering
2018 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Therapeutic proteins are becoming increasingly important. They are desirable, as they typically possess low adverse effects and higher specificity compared to the traditional, small molecule drugs. But they are also more complex and involve different intricate and expensive development and production processes. Through new technologies in protein and cell line development, more efficient and safer drugs can be readily available and at a lower cost. This thesis gives an overview of how protein therapeutics are developed and produced. It explores strategies to improve the efficacy and safety of protein drugs and how to improve production yields. In the present investigation, two papers present new methods for high-throughput cloning and site-directed mutagenesis using solid-phase immobilization of DNA fragments. These methods were designed to generate new drug candidates with swiftness and ease. Three papers show the development of a new cell line screening system that combines droplet microfluidics and the split-GFP reporter system. This combination allows for relative quantification of secreted recombinant proteins between individual cells and provides a tool for the selection of the best-producing clones for final production from a heterologous cell pool. The final paper explores the possibility to produce proteins at a higher cell density by examining how the metabolome and proteome of a perfusion bioreactor evolve as the cell density reaches exceptionally high levels. The consistent goal of all of these studies is to expedite the development and improve the production of therapeutic proteins, to assist the discovery of new drugs and to bring down production and development costs. Engineered proteins can be used to cure previously incurable diseases or give current medications a higher efficacy. Lower production and development costs can make the treatments available to more people.

Ort, förlag, år, upplaga, sidor
KTH Royal Institute of Technology, 2018
Serie
TRITA-CBH-FOU ; 2018:14
Nyckelord
Cell line development, therapeutic proteins, protein engineering, molecular cloning, mutagenesis, split-GFP
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-227195 (URN)978-91-7729-757-4 (ISBN)
Disputation
2018-05-31, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20180507

Tillgänglig från: 2018-05-07 Skapad: 2018-05-04 Senast uppdaterad: 2020-01-10Bibliografiskt granskad

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Hu, Francis JingxinLundqvist, MagnusUhlén, MathiasRockberg, Johan

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Hu, Francis JingxinLundqvist, MagnusUhlén, MathiasRockberg, Johan
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ProteinteknologiScience for Life Laboratory, SciLifeLabAlbanova VinnExcellence Center for Protein Technology, ProNova
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

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