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Development of an expression system for a dehydrogenase
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
2010 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

In recent years, biocatalytical steps in chemical synthesis are becoming increasingly important for economical and environmental-friendly production. In order to evaluate the use of enzymes in a process at Cambrex Karlskoga AB, an expression system was developed for a dehydrogenase. A synthetic gene was cloned into Escherichia coli DH5a cells, using the pTZ19R expression vector, as previously described in the literature. Protein expression was carried out at 25°C, 30°C and 37°C and results were measured using SDS-PAGE and activity assays. To improve expression, the gene was modified in three ways using PCR, yielding eight clones: It was inserted into the pSE420 expression vector, shortened to avoid inclusion body formation and a missing nucleotide was inserted into the sequence. A protocol for inclusion body screening was also developed. Finally, an assay for determining the kinetic constants of dehydrogenase was designed. It is concluded that further experiments must be done to obtain expression of the dehydrogenase and recommendations for additional work are given.

 

Abstract [sv]

Biokatalytiska processteg har de senaste åren blivit ett allt viktigare inslag i kemisk syntes för att åstadkomma ekonomisk och miljövänlig produktion. För att utvärdera användandet av enzymer i en process hos Cambrex Karlskoga AB utvecklades ett expressionssystem för ett dehydrogenas. En syntetisk gen klonades in i Escherichia coli DH5a och uttrycktes med hjälp av expressionsvektorn pTZ19R, som tidigare finns beskrivet i litteraturen. Proteinuttrycket utfördes vid 25°C, 30°C och 37°C och resultatet mättes med hjälp av SDS-PAGE och aktivitetsmätningar. Genen för dehydrogenaset modifierades på tre sätt, vilket gav upphov till åtta varianter. Genen fördes över till expressionsvektorn pSE420, kortades för att undvika bildning av inklusionskroppar och en nukleotid som fattades från gensekvensen återinfördes. Ett protokoll utarbetades även för undersökning av inklusionskroppar. Till sist sammanställdes en metod för att undersöka de kinetiska konstanterna hos dehydrogenaset. Slutsatsen av arbetet är att fortsatta studier måste utföras för att erhålla uttryck av dehydrogenaset och rekommendationer ges för framtida undersökningar.

 

Place, publisher, year, edition, pages
2010. , 36 p.
Keyword [en]
biocatalysis, protein expression, gene expression, inclusion bodies, SDS-PAGE, polymerase chain reaction, pTZ19R, pSE420
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-64344ISRN: LITH-IFM-A-EX--10/2235--SEOAI: oai:DiVA.org:liu-64344DiVA: diva2:389610
Presentation
2010-04-12, Linköping, 10:00 (Swedish)
Uppsok
Physics, Chemistry, Mathematics
Supervisors
Examiners
Available from: 2011-01-24 Created: 2011-01-19 Last updated: 2011-01-24Bibliographically approved

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CiteExportLink to record
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