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Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-7875-2822
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
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2015 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 7, article id e49Article in journal (Refereed) Published
Abstract [en]

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.

Place, publisher, year, edition, pages
2015. Vol. 43, no 7, article id e49
Keyword [en]
PCR Products, Restriction Enzymes, Magnetic Beads, In-Vitro, One-Pot, Protein, Polymerase, Expression, Construction, Proteomics
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-159278DOI: 10.1093/nar/gkv036ISI: 000354722500007PubMedID: 25618848Scopus ID: 2-s2.0-84961523206OAI: oai:DiVA.org:kth-159278DiVA, id: diva2:784050
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceNovo NordiskKnut and Alice Wallenberg FoundationVINNOVA
Note

QC 20150203

Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2018-05-04Bibliographically approved
In thesis
1. Targeted proteomics methods for protein quantification of human cells, tissues and blood
Open this publication in new window or tab >>Targeted proteomics methods for protein quantification of human cells, tissues and blood
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The common concept in this thesis was to adapt and develop quantitative mass spectrometric assays focusing on reagents originating from the Human Protein Atlas project to quantify proteins in human cell lines, tissues and blood. The work is based around stable isotope labeled protein fragment standards that each represent a small part of a human protein-coding gene. This thesis shows how they can be used in various formats to describe the protein landscape and be used to standardize mass spectrometry experiments. The first part of the thesis describes the use of antibodies in combination with heavy stable isotope labeled antigens to establish a semi-automated protocol for protein quantification of complex samples with fast analysis time  (Paper~I). Paper II introduces a semi-automated cloning protocol that can be used to selectively clone variants of recombinant proteins, and highlights the automation process that is necessary for large-scale proteomics endeavors. This paper also describes the technology that was used to clone all protein standards that are used in all of the included papers.

                     

The second part of the thesis includes papers that focus on the generation and application of antibody-free targeted mass spectrometry methods. Here, absolute protein copy numbers were determined across human cell lines and tissues (Paper III) and the protein data was correlated against transcriptomics data. Proteins were quantified to validate antibodies in a novel method that evaluates antibodies based on differential protein expression across multiple cell lines (Paper IV). Finally, a large-scale study was performed to generate targeted proteomics assays (Paper V) based on protein fragments. Here, assay coordinates were mapped for more than 10,000 human protein-coding genes and a subset of peptides was thereafter used to determine absolute protein levels of 49 proteins in human serum.

                     

In conclusion, this thesis describes the development of methods for protein quantification by targeted mass spectrometry and the use of recombinant protein fragment standards as the common denominator.

Place, publisher, year, edition, pages
Stockholm: Kungliga Tekniska högskolan, 2016. p. 90
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:16
Keyword
proteomics, mass spectrometry, protein quantification, stable isotope standard, parallel reaction monitoring, immuno-enrichment
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-193951 (URN)978-91-7729-153-4 (ISBN)
Public defence
2016-11-11, Gard-aulan, Folkhälsomyndigheten, Nobels väg 18, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20161013

Available from: 2016-10-13 Created: 2016-10-13 Last updated: 2016-10-14Bibliographically approved
2. Methods for cell line and protein engineering
Open this publication in new window or tab >>Methods for cell line and protein engineering
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2018
Series
TRITA-CBH-FOU ; 2018:14
Keyword
Cell line development, therapeutic proteins, protein engineering, molecular cloning, mutagenesis, split-GFP
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-227195 (URN)978-91-7729-757-4 (ISBN)
Public defence
2018-05-31, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20180507

Available from: 2018-05-07 Created: 2018-05-04 Last updated: 2018-05-08Bibliographically approved

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Lundqvist, MagnusHudson, Elton P.Tegel, HannaHolmberg, AndersUhlén, MathiasRockberg, Johan

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