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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, 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, 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: 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: 2017-12-05Bibliographically 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. 90 p.
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

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

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