A versatile bacterial expression vector based on the synthetic biology plasmid pSB1

Škrlj, Nives; Erčulj, Nina; Dolinar, Marko

doi:10.1016/j.pep.2008.10.019

Cited by 12 publications

(13 citation statements)

References 16 publications

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“…Liquid cultures were cultivated overnight at 37 • C on Luria-Bertani broth containing appropriate antibiotics. Bacterial biomass from Escherichia coli strain DH5␣ containing plasmid vector pMD204 (2345 bp) was obtained from the Faculty of Chemistry and Chemical Technology (University of Ljubljana, Slovenia) [20]. Bacteria were lysed using lysis procedure according to Smrekar et al [21].…”

Section: Cell Lysis Protocol and Plasmid Purificationmentioning

confidence: 99%

Sample displacement chromatography of plasmid DNA isoforms

Černigoj

Martinuč

Cardoso

et al. 2015

Journal of Chromatography A

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Section: Cell Lysis Protocol and Plasmid Purificationmentioning

confidence: 99%

Sample displacement chromatography of plasmid DNA isoforms

Černigoj

Martinuč

Cardoso

et al. 2015

Journal of Chromatography A

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“…To make cloning easier and faster, the novel expression vector pMD204 was used [15]. In vectors pMD204-HLL and pMD204-LLH, parts that constitute scFv are separated by unique restriction sites; therefore, each of them can be easily exchanged.…”

Section: Discussionmentioning

confidence: 99%

“…Cell samples were collected prior to and after IPTG induction for analysis of total bacterial proteins. Cell lysates and soluble and insoluble fractions were prepared as described [15]. Periplasmic fraction was isolated essentially as described [18].…”

Section: Assembly Of Scfvsmentioning

confidence: 99%

“…For V5B2-derived scFv production in E. coli, we used expression vector pMD204, especially designed for easy cloning of fusion segments connected by a linker [15]. Two constructs with opposing orientation of the light and heavy chain were prepared.…”

Section: Cloningmentioning

confidence: 99%

“…Single-chain Fvs, derived from mAb V5B2, were prepared in Escherichia coli periplasmic space using a tailor-made expression vector pMD204 [15]. This vector was developed specially for fusion protein construction and its bacterial production.…”

Section: Introductionmentioning

confidence: 99%

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Single-Chain Fv Antibody Fragments Retain Binding Properties of the Monoclonal Antibody Raised Against Peptide P1 of the Human Prion Protein

Škrlj

Šerbec

Dolinar

2009

Appl Biochem Biotechnol

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Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP(Sc)-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics.

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