Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Increased expression of therapeutic proteins by identification of 3'-UTRs from high expressing genes in CHO cells
Linköping University, Department of Physics, Chemistry and Biology.
2019 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Therapeutic proteins, a.k.a. biopharmaceuticals, are most commonly produced in expression systems derived from Chinese Hamstery Ovary (CHO) cells, thanks to great capacity of post-translational modifications like secretation, folding and glycosylation. The engineering of cells for regulation of protein expression has many options including knock-in and knock-out of genes, epigenetic studies or improvement of the expression casette of the protein of interest by e.g. promotor variants or modifications of the 5’ and 3’ untranslated region (UTR). The 3’-UTR is therefore a good optimization candidate for attempting to achieve increased expression of therapeutic proteins. The final aim of this study was to identify and design 3’-UTRs for improved expression of therapeutic proteins in HyClone™ CHO cells from GE Healthcare Bio-Sciences AB (GEHC). The impact goal is to increase the efficiency and lower the costs for pharmaceutical companies when producing biopharmaceuticals in the HyClone™ CHO cell line, leading to increased accessibility of monoclonal antibodies (mAbs) on the pharmaceutical market.

The study was initiated with bioinformatic analysis of the CHO cell transcriptome from a set of RNA-seq data of HyClone™ CHO to find high expressing, context independent genes. The 3’-UTRs from the best candidate genes were used for construction of plasmids for expression of a Fc-eGFP fusion protein. Nine selected 3’-UTRs were designed, synthesized and cloned into a parent plasmid (pGE0520) creating nine plasmid variants (pGE0523-531). The constructed plasmids were used for evaluation with site directed integration (SDI) into the HyClone™ CHO cell line and expression analysis were performed by flow cytometry and antibody titer measurements from cells with successfully integrated plasmid sorted by fluorescence-activated cell sorting (FACS).  

Result show a significant effect on protein expression when using different variants of 3’-UTRs. Two variants, pGE0524 and pGE0526, competing with the parent plasmid in expression levels and integration efficiency from SDI, making them candidates for further investigations against the parent plasmid. Results also show good correlation between flow cytometry data from pre- and post-sorting, which can make research for further 3’-UTRs more efficient by evaluations and prediction of expression levels before cell sorting.

Place, publisher, year, edition, pages
2019. , p. 41
Keywords [en]
Mammalian cell, CHO, E. coli, protein expression, plasmid, transformation, Fc-fusion protein, flow cytometry, FACS, transfection, mRNA, UTR, gene engineering
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:liu:diva-157593ISRN: LITH-IFM-A-EX--19/3660--SEOAI: oai:DiVA.org:liu-157593DiVA, id: diva2:1325235
External cooperation
GE Healthcare Bio-Sciences AB
Subject / course
Chemistry
Presentation
2019-06-10, Jordan/Fermi (Fysikhuset), Linköpings universitet, 581 83 Linköping, 16:00 (Swedish)
Examiners
Available from: 2019-06-17 Created: 2019-06-14 Last updated: 2019-06-17Bibliographically approved

Open Access in DiVA

MasterThesis_AlexanderWestlund(8608 kB)16 downloads
File information
File name FULLTEXT01.pdfFile size 8608 kBChecksum SHA-512
1a4193d5e9f75b9edb31185ff06786a58bb61cd435aa24a65dad4a46016727008bbef3828c682223646fedffa61e70ac215dfcc33d8cca10f52d637336b85d8c
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Westlund, Alexander
By organisation
Department of Physics, Chemistry and Biology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
Total: 16 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

urn-nbn

Altmetric score

urn-nbn
Total: 58 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf