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Bacterial Recombinant Expression by the Positively Regulated XylS/Pm Promoter System - Comparative, Genetic and Physiological Studies on Modulation of Protein Production Levels
Norwegian University of Science and Technology, Faculty of Natural Sciences and Technology, Department of Biotechnology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [no]

Økt kontroll over rekombinant proteinproduksjon i bakterier

Rekombinant proteinproduksjon i bakterier beskriver en forskningsaktivitet der man vil produsere nyttige proteiner til for eksempel medisinsk eller industriell bruk ved å innføre DNA fra en fremmed organisme. For at dette skal fungere er det ikke nok å lese av de såkallte rekombinante genene og lage de tilsvarende proteiner. Det må skje på en nøye regulert måte slik at alle delfunksjoner blir kvantitativt tilpasset hverandre. I praksis betyr dette at man bruker forskjellige genetiske kontrollelementer som påvirker transkripsjon eller translasjon.

For å teste effekten av ulike DNA sekvenser på proteinproduksjon brukte jeg blant annet fluorescerende proteiner som enkelt kan detekteres. Regulerbare promotere som først og fremst påvirker transkripsjon er ofte brukt for å kontrollere produksjon av et rekombinant protein. En sentral del av arbeidet mitt var å kartlegge egenskapene til ulike promotersystemer på en veldig systematisk måte.

En annen viktig kontrollregion er DNA region som koder for den 5’-utranslerte regionen (5’-UTR) i mRNA. Den ligger mellom promoter og gen og er spesiell fordi den påvirker både transkripsjon og translasjon. Nyutviklede dataprogrammer prøver å forutsi effekter utifra en 5’-UTR DNA sekvens eller til og med foreslår nye sekvenser med en forventet effekt (f.eks. høy proteinproduksjon). Men det har ikke vært godt samsvar mellom teori og praksis hittil. Jeg jobbet med å finne muligheter for å øke samsvaret mellom biologiske data og bioinformatiske beregninger. Først og fremst kunne jeg vise at det var mulig å få mer kontroll over proteinproduksjon ved å fysisk separere regioner innenfor 5’-UTR som påvirker transkripsjon fra regioner som påvirker translasjon. Så klarte jeg å finne DNA sekvenser som stimulerte de to prosessene hver for seg. Sammenlagt hadde disse en svært positiv, synergistisk effekt.

Mine studier økte forståelsen av de mest grunnleggende mekanismene bak kontroll av rekombinant proteinproduksjon for å styre prosessen på en mer kontrollert måte i fremtiden.

Abstract [en]

Genetically engineered bacteria, especially Escherichia coli, find applications in both university and industry-based research; one important purpose being heterologous production of proteins. Experiences from empiric genetic engineering strategies in pursuit of maximizing or, generally speaking, modulating protein production levels have demonstrated that results are often unpredictable. Even after several decades of research in this field, the complex interplay of the many different genetic and physiological parameters that affect protein production levels are still not fully understood. Among the various expression vectors, mini-RK2 replicons containing the positively regulated xylS/Pm system can be used in E. coli and other Gram-negative bacteria. Using recombinant E. coli strains harboring these vectors as a basis, the influence of different genetic and physiological parameters on recombinant gene expression was systematically compared during this PhD project.

The first parameter studied was how different regulated promoter systems influence expression. Interestingly, a survey of the scientific literature indicated that the performance of commonly used expression systems such as LacI/PT7lac, LacI/Ptrc, and AraC/PBAD, (and also XylS/Pm) had to a very limited extent been properly and systematically compared to each other. Such a comparison was selected as one objective of this PhD project. Given a common vector backbone and expression system insertion points, this comparison made it possible to understand the influence of the systems on production of different selected proteins. This study confirmed that no system was superior to meet all requirements an ideal system should have. However, it was possible to recommend certain systems for different expression purposes. The LacI/PT7lac system, for example, was still best suited for achieving most total production judged by the vast amount of accumulated transcript and total recombinant protein. As for production of soluble (and active) protein, however, use of a variant of XylS/Pm (XylS/Pm ML1-17) or AraC/PBAD were more beneficial for some proteins. The plasmid vector tools developed for this study may be used in future studies to analyze limiting factors for production of in principle any specific protein, including production in an active form.

The second genetic parameter included in the systematic studies was the DNA region corresponding to the 5′-UTR of mRNA. My findings demonstrate that 5′-UTR DNA sequences with strong RBS (like the PT7lac UTR), could enhance protein (here: ß-lactamase) production from XylS/Pm (7-fold) compared to the Pm 5′-UTR. Still, Pm 5′-UTR DNA sequences that were obtained by combinatorial mutagenesis and screening approaches gave rise to even higher protein amounts (up to 20-fold). Also, the relative changes could not be predicted using current sequence analysis tools. Prior to this study, it was already shown that a 5′-UTR DNA region plays a key role in expression due to its involvement in transcription, translation and transcript stability. Therefore, it was hypothesized that one cannot study the effect of 5′-UTR DNA regions on protein production based on parameters influencing translation only (e.g. length of a Shine-Dalgarno sequence). To determine sequence features that influence transcription and to distinguish them from features influencing translation, combinatorial mutagenesis and screening using two efficient vector tools was applied. The new 5′-UTR DNA regions identified with these tools led to primarily stimulated transcript accumulation or protein production indeed, but it was not possible to identify positional hot-spots for mutations that specifically influenced either process. It was however possible to combine a 5′-UTR DNA region carrying mutations that primarily stimulated transcript accumulation with a 5′-UTR DNA region whose mutations primarily stimulated translation The total improvement achieved by this strategy was an impressive 170- fold compared to the native 5′-UTR. It can also be possible to adjust this 5′-UTR DNA region to other promoters and to other coding regions in the future. Hopefully, this strategy will enable more rational design in recombinant protein expression.

Among the remaining parameters that were varied, it was not surprisingly growth temperature, inducer concentration and plasmid (and indirectly gene) copy number that influenced recombinant protein production. This latter parameter also influenced culture heterogeneity at the single-cell level.

The last parameter that this work focused on was the qualitative influence of the expression host on the final product. Certain proteins are prone to be problematic for functional expression in E. coli even independent of the expression levels, the growth conditions or the protein engineering strategies. This was demonstrated by the example of the two difficult-to-express Norwegian Salmonid alphavirus E1 and E2 capsid proteins. Therefore, exploring the use of alternative bacterial hosts was considered; Pseudomonas putida, the organism XylS/Pm originates from, as well as cold-adapted members of the genus Pseudomonas. Vectors developed in this project were suitable to be directly transferred to these hosts; and at least for one protein (mCherry), even higher protein production levels could be achieved in P. putida compared to E. coli. The cold-adapted Pseudomonas strains also showed potential to be used as expression hosts for certain proteins, however, slow growth and low expression levels demonstrated the need to engineer these novel hosts further in the future.

The findings presented in this work not only expand our current understanding of gene expression in general, but also help to approach the goal to optimize bacterial recombinant protein production in a more rational manner than is currently possible.

Place, publisher, year, edition, pages
NTNU, 2014.
Doctoral theses at NTNU, ISSN 1503-8181 ; 2014:114
National Category
URN: urn:nbn:no:ntnu:diva-24984ISBN: 978-82-326-0152-3 (printed ver.)ISBN: 978-82-326-0153-0 (electronic ver.)OAI: diva2:727096
Public defence
2014-04-25, 13:15
Available from: 2014-06-19 Created: 2014-06-19 Last updated: 2014-06-19Bibliographically approved
List of papers
1. A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli
Open this publication in new window or tab >>A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli
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2013 (English)In: Microbial Cell Factories, ISSN 1475-2859, Vol. 12, 26- p.Article in journal (Refereed) Published
Abstract [en]

Background: Production of recombinant proteins in bacteria for academic and commercial purposes is a well established field; however the outcomes of process developments for specific proteins are still often unpredictable. One reason is the limited understanding of the performance of expression cassettes relative to each other due to different genetic contexts. Here we report the results of a systematic study aiming at exclusively comparing commonly used regulator/promoter systems by standardizing the designs of the replicon backbones. Results: The vectors used in this study are based on either the RK2- or the pMB1-origin of replication and contain the regulator/promoter regions of XylS/Pm (wild-type), XylS/Pm ML1-17 (a Pm variant), LacI/P-T7lac, LacI/P-trc and AraC/P-BAD to control expression of different proteins with various origins. Generally and not unexpected high expression levels correlate with high replicon copy number and the LacI/P-T7lac system generates more transcript than all the four other cassettes. However, this transcriptional feature does not always lead to a correspondingly more efficient protein production, particularly if protein functionality is considered. In most cases the XylS/Pm ML1-17 and LacI/P-T7lac systems gave rise to the highest amounts of functional protein production, and the XylS/Pm ML1-17 is the most flexible in the sense that it does not require any specific features of the host. The AraC/P-BAD system is very good with respect to tightness, and a commonly used bioinformatics prediction tool (RBS calculator) suggested that it has the most translation-efficient UTR. Expression was also studied by flow cytometry in individual cells, and the results indicate that cell to cell heterogeneity is very relevant for understanding protein production at the population level. Conclusions: The choice of expression system needs to be evaluated for each specific case, but we believe that the standardized vectors developed for this study can be used to more easily identify the nature of case-specific bottlenecks. By then taking into account the relevant characteristics of each expression cassette it will be easier to make the best choice with respect to the goal of achieving high levels of protein expression in functional or nonfunctional form.

Recombinant expression, Regulator/promoter systems, XylS/Pm, XylS/Pm ML1-17, LacI/P-T7lac, LacI/P-trc, AraC/P-BAD, Systematic comparison
urn:nbn:no:ntnu:diva-24981 (URN)10.1186/1475-2859-12-26 (DOI)000317311500001 ()

© 2013 Balzer et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Available from: 2014-06-19 Created: 2014-06-19 Last updated: 2014-06-19Bibliographically approved
2. Improving bacterial recombinant gene expression by separate optimization and combination of two functional parts of 5’-UTR sequences.
Open this publication in new window or tab >>Improving bacterial recombinant gene expression by separate optimization and combination of two functional parts of 5’-UTR sequences.
(English)Manuscript (preprint) (Other academic)
urn:nbn:no:ntnu:diva-24983 (URN)
Available from: 2014-06-19 Created: 2014-06-19 Last updated: 2014-06-19Bibliographically approved
3. High production of recombinant Norwegian salmonid alphavirus E1 and E2 proteins in Escherichia coli by fusion to secretion signal sequences and removal of hydrophobic domains
Open this publication in new window or tab >>High production of recombinant Norwegian salmonid alphavirus E1 and E2 proteins in Escherichia coli by fusion to secretion signal sequences and removal of hydrophobic domains
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2013 (English)In: BIOTECHNOL BIOPROC E, ISSN 1226-8372, Vol. 18, no 4, 742-750 p.Article in journal (Refereed) Published
Abstract [en]

The Norwegian salmonid alphavirus (NSAV) infects farmed Atlantic salmon and rainbow trout, causes pancreas disease and leads to economic losses and fish health issues for the aquaculture. Vaccines are available, but recurring infection outbreaks at Norwegian fish farms have led to endeavours in finding solutions for increased prevention. The NSAV E1 and E2 envelope proteins are potential targets for production of recombinant subunit vaccines and for generation of antibodies for diagnostics. Efficient expression of target proteins is necessary for these applications, and here we present a new strategy for expressing this kinds of viral proteins. We show that 5'-terminal fusion of signal sequences OmpA and CSP to the e1 and e2 genes and removal of the C-terminal hydrophobic interaction and transmembrane domains of E1 and E2 leads to significantly increased expression levels. Recombinant Escherichia coli strains for high-level production of E1 and E2 harbouring these modifications were established using the inducible XylS/Pm expression cassette. Furthermore, reduction of temperature to 16A degrees C after induction leads to 4-fold increase in production for E1, and under high-cell-density cultivations we obtained production levels up to 2.3 g/L. We also show that these proteins can be purified from inclusion bodies by affinity chromatography. This demonstrates the present approach as promising for large scale production of such viral proteins.

recombinant vaccines, recombinant gene expression, XylS/Pm, high-cell-density cultivations, signal sequences
urn:nbn:no:ntnu:diva-24982 (URN)10.1007/s12257-013-0085-y (DOI)000323244600016 ()
Available from: 2014-06-19 Created: 2014-06-19 Last updated: 2014-06-19Bibliographically approved

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