Helmut Merk

Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system.

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.

Cell-free synthesis of functional and endotoxin-free antibody Fab fragments by translocation into microsomes

A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase. The applicability of the system is demonstrated by the synthesis of anti-lysozyme and anti-CD4 Fab antibody fragments yielding approximately 10 µg Fab per milliliter reaction mixture. The lack of endotoxins in this system is a prerequisite that synthesized Fab can be applied directly using whole synthesis reactions in cell-based assays that are sensitive to this substance class. Moreover, the system is compatible with PCR-generated linear templates enabling automated generation of antibody fragments in a high-throughput manner, and facilitating its application for screening and validation purposes.

Chapter 2 In Vitro Synthesis of Posttranslationally Modified Membrane Proteins

Publisher Summary Membrane proteins have become an important focus of the current efforts in structural and functional genomics and the rapid progress of various genome sequencing projects has greatly accelerated the discovery of novel genes encoding membrane proteins. In contrast, the molecular analysis of membrane proteins lags far behind that of cytosolic soluble proteins. Preparing high quality samples of functionally folded proteins represents a major bottleneck that restricts further structural and functional studies. Cell-free protein expression systems, in particular those of eukaryotic origin, have recently been developed as promising tools for the rapid and efficient production of a wide variety of membrane proteins. A large number of these proteins, however, require posttranslational modifications for optimum function. Several membrane proteins have been expressed in vivo to date, most of them being functionally, antigenically, and immunogenically similar to their authentic counterparts. This is mainly because of the properties of cultured eukaryotic cells, which are able to carry out many types of posttranslational modifications, such as the addition of N- and O-linked oligosaccharides, but also palmitoylation, myristylation, and phosphorylation.

Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems

Eukaryotic cell-free systems based on wheat germ and Spodoptera frugiperda insect cells were equipped with an orthogonal amber suppressor tRNA-synthetase pair to synthesize proteins with a site-specifically incorporated p-azido-l-phenylalanine residue in order to provide their chemoselective fluorescence labeling with azide-reactive dyes by Staudinger ligation. The specificity of incorporation and bioorthogonality of labeling within complex reaction mixtures was shown by means of translation and fluorescence detection of two model proteins: β-glucuronidase and erythropoietin. The latter contained the azido amino acid in proximity to a signal peptide for membrane translocation into endogenous microsomal vesicles of the insect cell-based system. The results indicate a stoichiometric incorporation of the azido amino acid at the desired position within the proteins. Moreover, the compatibility of cotranslational protein translocation, including glycosylation and amber suppression-based incorporation of p-azido-l-phenylalanine within a cell-free system, is demonstrated. The presented approach should be particularly useful for providing eukaryotic and membrane-associated proteins for investigation by fluorescence-based techniques.

A novel in vitro translation system based on insect cells

Background Various genome sequencing projects are identifying many new protein sequences but it is key to attribute functions to these proteins. A huge number of proteins have been expressed in vivo to date, most of them being functionally, antigenically and immunogenically similar to their authentic counterparts. This is mainly due to the properties of cultured eukaryotic cells, which are able to carry out many types of posttranslational modifications such as addition of Nand O-linked oligosaccharides, but also palmitoylation, myristylation and phosphorylation.

Expression-PCR: from Gene Pools to Purified Proteins Within 1 Day

Prokaryotic cell-free protein biosynthesis has become a well-established and powerful tool for the synthesis of gene products and the analysis of gene expression. One of the main advantages of this methodology is the very short time which is needed for the expression of proteins in sufficient amounts for structure and function determination. One indispensable prerequisite to successful protein synthesis is the nature of the template, which needs to contain the appropriate regulatory elements for the reaction besides the coding region. However, template construction and preparation remain time-consuming because cloning procedures, bacterial amplification and subsequent isolation from bacteria are necessary in the usual systems. To overcome these limitations, the methodology of expression-PCR (E-PCR) was developed, in which the coding region of any intronless gene is completed with the necessary regulatory elements in the 5’ and 3’ untranslated regions via polymerase chain reaction (PCR). The obtained PCR products could be used directly as templates first for eukaryotic cell-free protein synthesis [3, 6, 7, 15, 17]. Templates designed for use in the more productive prokaryotic protein synthesis system (i.e., E. coli) need more regulatory sequence information than those for use in eukaryotic systems. The introduction of these sequences normally requires very long primers which can cause problems in the PCR. For this reason a successful and universal expression-PCR in the prokaryotic system is difficult [9, 10, 11, 13, 14].

Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing.

Abstract G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes. We provide a detailed study on the production efficiencies and quality of G protein-coupled receptors, Fab fragments and other proteins synthesized in insect cell lysates containing endogenous microsomes. Effects of different reaction kinetics, redox conditions and sample preparations on the specific activities of synthesized proteins have been analyzed. The extent of glycosylation, membrane translocation and percentages of ligand binding active fractions of synthesized protein samples have been determined. We provide strong evidence that membrane insertion of integral membrane proteins can represent a prime limiting factor for their preparative scale in vitro production. Improved expression protocols resulting into higher production rates yielded more active protein in case of Fab fragments, but not in case of the human endothelin B receptor.

Completion of Proteomic Data Sets by Kd Measurement Using Cell-Free Synthesis of Site-Specifically Labeled Proteins

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.

Recombinant protein production in cell-free systems: strategies for improving yield and functionality

Background Despite the widespread use of CHO cells for the production of many industrially relevant biopharmaceuticals, this system is poorly understood on the genetic level and mainly relies on empirical procedures, due to the lack of adequate sequence information. In order to advance its overall performance, we successfully tested the applicability of a cross-species microarray approach, for investigating CHO specific transcription profiles [1]. In the present study we show expression signatures of individual recombinant CHO clones which correlate with their associated phenotype.

Towards Improved Applications of Cell-Free Protein Biosynthesis - The Influence of mRNA Structure and Suppressor tRNAS on the Efficiency of the System

The cell-free protein biosynthesis has the potential to become a powerful technology for the biochemical research in particular in the determination of the structure and function of proteins. The number of possible applications is rising with the obtainable yields and with the expanded feasibility of introducing modified amino acids into proteins. Here we describe the influence of two RNA translation components, the mRNA and the suppressor tRNA, on the efficiency of protein biosynthesis.