Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteinsReport as inadecuate




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BMC Biotechnology

, 12:65

Protein and enzyme technology

Abstract

BackgroundConventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins FbFPs are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large 58% of the size of GFP.

ResultsAs an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody scFv Ki-4scFv and the anti-MucI single-chain fragment variable M12scFv. During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal was increased. Antibodies with W-tags generated stronger signals than the untagged construct.

ConclusionsOur low-molecular-weight W-tags can be used to monitor the production of antibody fragments on-line. The binding specificity of the recombinant fusion protein is not affected, even though the binding activity decreases slightly with increasing number of tryptophan residues in the W-tags. Thus, the newly designed W-tags offer a versatile and generally applicable alternative to current fluorescent fusion tags.

KeywordsTryptophan tagOn-line monitoringMicrotiter plateFluorescence measurementEscherichia coli protein expressionSmall scale fermentationAbbreviationsa.uArbitrary units

DOTDissolved oxygen tension

ECExpression control

EDTAEthylene-diamine-tetraacetic acid

FbFPFMN-based fluorescent protein

FMNFlavin mononucleotides

GFPGreen Fluorescent Protein

IPTGIsopropyl -D-1-thiogalactopyranoside

mABMonoclonal full length antibody

MFIMean fluorescence intensity

MTPMicrotiter plate

NCNegative control

ODOptical density

scFvSingle-chain fragment variable

WTryptophan one letter code for amino acid

YFPYellow Fluorescent Protein.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-12-65 contains supplementary material, which is available to authorized users.

Eva-Maria Siepert, Esther Gartz contributed equally to this work.

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Author: Eva-MariaSiepert - EstherGartz - MehmetKemalTur - HeinrichDelbrck - StefanBarth - JochenBchs

Source: https://link.springer.com/







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