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Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.ORCID-id: 0000-0002-7875-2822
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
Vise andre og tillknytning
2015 (engelsk)Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, nr 7, artikkel-id e49Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2015. Vol. 43, nr 7, artikkel-id e49
Emneord [en]
PCR Products, Restriction Enzymes, Magnetic Beads, In-Vitro, One-Pot, Protein, Polymerase, Expression, Construction, Proteomics
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
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
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceNovo NordiskKnut and Alice Wallenberg FoundationVINNOVA
Merknad

QC 20150203

Tilgjengelig fra: 2015-01-28 Laget: 2015-01-28 Sist oppdatert: 2020-01-30bibliografisk kontrollert
Inngår i avhandling
1. Targeted proteomics methods for protein quantification of human cells, tissues and blood
Åpne denne publikasjonen i ny fane eller vindu >>Targeted proteomics methods for protein quantification of human cells, tissues and blood
2016 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Kungliga Tekniska högskolan, 2016. s. 90
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:16
Emneord
proteomics, mass spectrometry, protein quantification, stable isotope standard, parallel reaction monitoring, immuno-enrichment
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-193951 (URN)978-91-7729-153-4 (ISBN)
Disputas
2016-11-11, Gard-aulan, Folkhälsomyndigheten, Nobels väg 18, Solna, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 20161013

Tilgjengelig fra: 2016-10-13 Laget: 2016-10-13 Sist oppdatert: 2020-01-10bibliografisk kontrollert
2. Methods for cell line and protein engineering
Åpne denne publikasjonen i ny fane eller vindu >>Methods for cell line and protein engineering
2018 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
KTH Royal Institute of Technology, 2018
Serie
TRITA-CBH-FOU ; 2018:14
Emneord
Cell line development, therapeutic proteins, protein engineering, molecular cloning, mutagenesis, split-GFP
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-227195 (URN)978-91-7729-757-4 (ISBN)
Disputas
2018-05-31, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 20180507

Tilgjengelig fra: 2018-05-07 Laget: 2018-05-04 Sist oppdatert: 2020-01-10bibliografisk kontrollert

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