Michael Gerrits

Gene optimization mechanisms: a multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli.

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.

Site-specific PEGylation of proteins by a Staudinger-phosphite reaction

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.

Synthesis of membrane proteins in eukaryotic cell‐free systems

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.

Fast Biosynthesis of GFP Molecules: A Single‐Molecule Fluorescence Study

Its not easy being green: Real-time visualization of labeled ribosomes and de novo synthesized green fluorescent protein molecules using single-molecule-sensitive fluorescence microscopy demonstrates that the mutant GFPem is produced with a characteristic time of five minutes. Fluorescence of the fastest GFP molecules appears within one minute (see picture).

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.

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.

Chemoselective Bioconjugation of Triazole Phosphonites in Aqueous Media

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.