RiNA GmbH

Berlin, Germany

RiNA GmbH

Berlin, Germany
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Von Hacht A.,TU Berlin | Seifert O.,University of Stuttgart | Menger M.,RiNA GmbH | Schutze T.,TU Berlin | And 7 more authors.
Nucleic Acids Research | Year: 2014

Guanine quadruplex (G-quadruplex) motifs in the 5′ untranslated region (5′-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophysical characterization and the inhibitory activity on translation was shown by luciferase reporter assays. In experiments with whole cell extracts from different eukaryotic cell lines, G-quadruplex-binding proteins were isolated by pull-down assays and subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The binding partners of the RNA G-quadruplexes we discovered included several heterogenous nuclear ribonucleoproteins, ribosomal proteins, and splicing factors, as well as other proteins that have previously not been described to interact with nucleic acids. While most of the proteins were specific for either of the investigated G-quadruplexes, some of them bound to both motifs. Selected candidate proteins were subsequently produced by recombinant expression and dissociation constants for the interaction between the proteins and RNA G-quadruplexes in the low nanomolar range were determined by surface plasmon resonance spectroscopy. The present study may thus help to increase our understanding of the mechanisms by which G-quadruplexes regulate translation. © 2014 The Author(s) 2014.


Uzagare M.C.,Albert Ludwigs University of Freiburg | Claussnitzer I.,RiNA GmbH | Gerrits M.,RiNA GmbH | Bannwarth W.,Albert Ludwigs University of Freiburg
Organic and Biomolecular Chemistry | Year: 2012

The bioorthogonal and chemoselective fluorescence labelling of several cell-free synthesized proteins containing a site-specifically incorporated azido amino acid was possible using different alkyne-functionalized Ru(ii) bathophenanthroline complexes. We were able to achieve a selective labelling even in complex mixtures of proteins despite the fact that ruthenium dyes normally show a high tendency for unspecific interactions with proteins and are commonly used for total staining of proteins. Since the employed Ru complexes are extremely robust, photo-stable and highly sensitive, the approach should be applicable to the production of labelled proteins for single molecule spectroscopy and fluorescence-based interaction studies. © The Royal Society of Chemistry 2012.


Vallee M.R.J.,Leibniz Institute for Molecular Pharmacology | Majkut P.,Leibniz Institute for Molecular Pharmacology | Krause D.,Leibniz Institute for Molecular Pharmacology | Gerrits M.,RiNA GmbH | And 2 more authors.
Chemistry - A European Journal | Year: 2015

Readily accessible and versatile phosphonite building blocks with improved stability against hydrolysis were used for the efficient metal-free functionalization of peptides and proteins in aqueous buffers at low micromolar concentrations. The application of this protocol to the immobilization of a Rasa1-SH2 domain revealed high binding affinity to the human T-cell protein ADAP and supports the applicability of triazole phosphonites for protein modifications without harming their function. © 2015 Wiley-VCH Verlag GmbH & Co. KGaA.


Majkut P.,Leibniz Institute for Molecular Pharmacology | Claussnitzer I.,RiNA GmbH | Merk H.,RiNA GmbH | Freund C.,Free University of Berlin | And 3 more authors.
PLoS ONE | Year: 2013

The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level. © 2013 Majkut et al.


Serwa R.,Free University of Berlin | Majkut P.,Free University of Berlin | Horstmann B.,Free University of Berlin | Swiecicki J.-M.,Free University of Berlin | And 3 more authors.
Chemical Science | Year: 2010

Current protocols in protein bioengineering allow the site-specific incorporation of chemical reporter moieties. Subsequently, these functional groups can be chemoselectively transformed to decorate proteins with charged and oversized functional units. Based on our recent report on the chemoselective reaction of azides with phosphites, we now apply the Staudinger-phosphite reaction to an efficient and metal-free PEGylation of an azide-containing protein with symmetrical phosphites. Thereby, two types of branched oligoethylene glycol scaffolds are generated, which deliver either a stable or light-cleavable protein-PEG conjugate. Furthermore, we demonstrate that the Staudinger-phosphite reaction is an efficient transformation in both aqueous media as well as in a highly crowded bacterial cell lysate. © 2010 The Royal Society of Chemistry.


Stech M.,Fraunhofer Institute for Biomedical Engineering | Merk H.,RiNA GmbH | Schenk J.A.,UP Transfer GmbH | Schenk J.A.,Hybrotec GmbH | And 7 more authors.
Journal of Biotechnology | Year: 2013

Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner. © 2012 Elsevier B.V.


Sachse R.,Fraunhofer Institute for Biomedical Engineering | Wustenhagen D.,Fraunhofer Institute for Biomedical Engineering | Samalikova M.,Fraunhofer Institute for Biomedical Engineering | Gerrits M.,RiNA GmbH | And 3 more authors.
Engineering in Life Sciences | Year: 2013

Cell-free protein synthesis (CFPS) is a valuable method for the fast expression of difficult-to-express proteins as well as posttranslationally modified proteins. Since cell-free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell-free translation systems are capable to provide high molecular weight membrane proteins, fluorescent-labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Maertens B.,Qiagen | Spriestersbach A.,Qiagen | Von Groll U.,Qiagen | Roth U.,Qiagen | And 8 more authors.
Protein Science | Year: 2010

The genetic code is universal, but recombinant protein expression in heterologous systems is often hampered by divergent codon usage. Here, we demonstrate that reprogramming by standardized multi-parameter gene optimization software and de novo gene synthesis is a suitable general strategy to improve heterologous protein expression. This study compares expression levels of 94 full-length human wt and sequence-optimized genes coding for pharmaceutically important proteins such as kinases and membrane proteins in E. coli. Fluorescence-based quantification revealed increased protein yields for 70% of in vivo expressed optimized genes compared to the wt DNA sequences and also resulted in increased amounts of protein that can be purified. The improvement in transgene expression correlated with higher mRNA levels in our analyzed examples. In all cases tested, expression levels using wt genes in tRNA-supplemented bacterial strains were outperformed by optimized genes expressed in non-supplemented host cells. Published by Wiley-Blackwell. © 2010 The Protein Society.


Majkut P.,Free University of Berlin | Bohrsch V.,Free University of Berlin | Bohrsch V.,Berlin Technical University of Applied Sciences | Serwa R.,Free University of Berlin | And 2 more authors.
Methods in Molecular Biology | Year: 2012

Chemoselective reactions are important tools for the modification of peptides and proteins. Thereby the modification is desired to be site specific and bioorthogonal. Here we describe the site-specific modification of azido-proteins via a Staudinger-type phosphite ligation. The reaction was carried out in aqueous system on proteins containing p-azido-phenylalanine in a single position introduced by the amber codon technique. A selective introduction of branched polyethylene scaffolds can be achieved with the application of the methodology reported herein.


PubMed | University of Stuttgart, Goethe University Frankfurt, Free University of Berlin, Max Planck Institute of Colloids and Interfaces and 2 more.
Type: Journal Article | Journal: Nucleic acids research | Year: 2014

Guanine quadruplex (G-quadruplex) motifs in the 5 untranslated region (5-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophysical characterization and the inhibitory activity on translation was shown by luciferase reporter assays. In experiments with whole cell extracts from different eukaryotic cell lines, G-quadruplex-binding proteins were isolated by pull-down assays and subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The binding partners of the RNA G-quadruplexes we discovered included several heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and splicing factors, as well as other proteins that have previously not been described to interact with nucleic acids. While most of the proteins were specific for either of the investigated G-quadruplexes, some of them bound to both motifs. Selected candidate proteins were subsequently produced by recombinant expression and dissociation constants for the interaction between the proteins and RNA G-quadruplexes in the low nanomolar range were determined by surface plasmon resonance spectroscopy. The present study may thus help to increase our understanding of the mechanisms by which G-quadruplexes regulate translation.

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