Tagging of Escherichia coli proteins with new cassettes allowing in vivo systematic fluorescent and luminescent detection, and purification from physiological expression levels

Abstract

We designed cassettes allowing the systematic fusion of fluorescent or luminescent proteins preceded by the calmodulin binding peptide tag to the C-terminus of Escherichia coli proteins. The chromosomal insertion, and thus physiological expression level of these fusions, permits the study of protein localization by fluorescent microscopy and protein quantification, in vivo and dynamically in diverse conditions. Furthermore, the calmodulin binding peptide tag allows standard detection, affinity purification, and co-purification experiments. These cassettes are therefore very valuable for the versatility of experiments they make available for a given strain, from biochemistry to dynamic and in vivo studies.

Cross talk between ABC transporter m RNA s via a target m RNA ‐derived sponge of the G cv B small RNA

“…To integrate oppA and gltI translational fusions into Salmonella chromosome, oppA 16aa ‐ sfgfp and gltI 16aa ‐ sfgfp were amplified with primer pairs JVO‐10629/JVO‐9762 and JVO‐10631/JVO‐10688 using pDP124 (pCBP derivative replaced with sfgfp ) (Wahl et al , ) as a template and were recombined by lambda Red system (Datsenko & Wanner, ). To omit the downstream terminator sequence and Km resistance gene, the C‐terminal 23 aa of the gltI sequence was fused with sfgfp by scar‐less mutagenesis (Blank et al , ).…”

Characterization of the RNase R association with ribosomes

“…Strains with fusion proteins were prepared using lambda red recombination method as described [16]. Plasmids were used as a template for the integration cassettes as described [25]. …”

A universal immuno-PCR platform for comparative and ultrasensitive quantification of dual affinity-tagged proteins in complex matrices

“…Indeed, SrtA-GFP-HA was detected with a similar LOD and dynamic range as for GFP-HA in serum, confirming the robustness of the platform. Affinity-tagged proteins have previously been used for protein detection in a diverse range of circumstances, such as biomarker library screening in different eukaryotic organisms, 3,8,14 tumourimplicated cytokine secretion profiling, 13 and expression and localization of tagged proteins in CSF of mice. 10,28 Universal methods for the comparative quantification of different proteins present in low abundance in various complex matrices are nevertheless rare.…”