Change search
ReferencesLink to record
Permanent link

Direct link
Recombinant Enzymes in Pyrosequencing Technology
KTH, Superseded Departments, Biotechnology.
2004 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Pyrosequencing is a DNA sequencing method based on thedetection of released pyrophosphate (PPi) during DNA synthesis.In a cascade of enzymatic reactions, visible light isgenerated, which is proportional to the number of nucleotidesincorporated into the DNA template. When dNTP(s) areincorporated into the DNA template, inorganic PPi is released.The released PPi is converted to ATP by ATP sulfurylase, whichprovides the energy to luciferase to oxidize luciferin andgenerate light. The excess of dNTP(s) and the ATP produced areremoved by the nucleotide degrading enzyme apyrase.

The commercially available enzymes, isolated from nativesources, show batch-tobatch variations in activity and quality,which decrease the efficiency of the Pyrosequencing reaction.Therefore, the aim of the research presented in this thesis wasto develop methods to recombinantly produce the enzymes used inthe Pyrosequencing method. Production of the nucleotidedegrading enzyme apyrase by Pichia pastoris expression system,both in small-scale and in an optimized large-scale bioreactor,is described. ATP sulfurylase, the second enzyme in thePyrosequencing reaction, was produced inEscherichia coli. The protein was purified and utilizedin the Pyrosequencing method. Problems associated with enzymecontamination (NDP kinase) and batch-to-batch variations wereeliminated by the use of the recombinant ATP sulfurylase.

As a first step towards sequencing on chip-format,SSB-(single-strand DNA binding protein)-luciferase and KlenowDNA polymerase-luciferase fusion proteins were generated inorder to immobilize the luciferase onto the DNA template.

The application field for the Pyrosequencing technology wasexpanded by introduction of a new method for clone checking anda new method for template preparation prior the Pyrosequencingreaction.

Keywords:apyrase, Pyrosequencing technology, Zbasictag fusion, luciferase, ATP sulfurylase, dsDNAsequencing, clone checking, Klenow-luciferase, SSB-luciferase,Pichia pastoris, Echerichia coli.

Place, publisher, year, edition, pages
Stockholm: Bioteknologi , 2004. , xii, 66 p.
Keyword [en]
apyrase, pyrosequencing, pichia pastoris, protein expression, fusion protein, luciferase, DNA template, clone checking, DNA polymerase
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-3765ISBN: 91-7283-756-XOAI: diva2:9615
Public defence
2004-05-28, 00:00
Available from: 2004-05-27 Created: 2004-05-27 Last updated: 2012-03-22Bibliographically approved

Open Access in DiVA

fulltext(9901 kB)1567 downloads
File information
File name FULLTEXT01.pdfFile size 9901 kBChecksum MD5
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Nourizad, Nader
By organisation
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 1567 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

Total: 482 hits
ReferencesLink to record
Permanent link

Direct link