A general method for the covalent labeling of fusion proteins with small molecules in vivo

Characterizing the movement, interactions, and chem. microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic mols. capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small mol. to a protein, the formation of stable complexes between biarsenical compds. and peptides contg. cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.

The Use of Valproic Acid in Transient Gene Expression with HEK-293E Cells

Divor Kiseljak

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins. Recently, valproic acid (VPA), a well known histone deacetylase inhibitor, has been suggested for the use in cell cultures to enhance recombinant protein expression. However, little was known about protein expression in HEK-293E cells under VPA treatment. Therefore, the first aim of this thesis was to describe the effects of VPA on protein expression by TGE in HEK-293E cells. The addition of VPA induced a dose-dependent increase in protein production. Under the optimal VPA concentration (3.75 mM) IgG volumetric productivity increased over 8-fold compared to the control. This was achieved through improvements in both integral viable cell density (IVCD) and cell specific productivity. The IVCD was improved by the VPA-induced cell growth arrest resulting in longer culture duration and extended protein production phase. The enhancement in specific productivity in VPA-treated cells was a consequence of higher cellular pDNA copy number and improved pDNA utilization that led to higher transgene mRNA levels. The study on limitations governing VPA-based TGE protein expression in HEK-293E cells revealed the bottlenecks at the transcriptional and translational levels, however the limitation did not appear to be at the level of protein folding or assembly for the model IgG antibody studied. In addition, the effect of VPA was tested on the expression of different recombinant monoclonal antibodies and Fc-fusion proteins. The results showed that VPA addition did not always significantly improve protein titers. Since the optimal VPA concentration was dependent on both the recombinant protein being expressed and the amount of coding pDNA used for transfection, we recommend evaluating the suitability of VPA addition and the optimization of VPA concentration in order to find the optimal conditions for TGE protein expression. We believe that this work will contribute to the design of more rational, robust, and successful expression platforms for recombinant protein production in HEK-293E cells. One of the major contributors to the cost of TGE process at large scale is the considerable amount of pDNA required for transfection. Therefore, the second aim of III this thesis was to determine a cost-effective method to reduce the pDNA amount. We showed that a part of the coding pDNA can be replaced with nonspecific (filler) DNA with minimal loss in volumetric productivity. The studies revealed that the presence of filler DNA allowed the coding pDNA to be distributed over a larger number of DNA-PEI complexes. This resulted in a higher percentage of transfected cells and may favor the better utilization of the coding pDNA by the cell’s transcription machinery. The use of filler DNA as a part of defined fed-batch process (based on the addition of glucose and plant derived protein hydrolysates) resulted in 25 fold reduction in coding pDNA amount requirement while maintaining volumetric protein productivity over 1 g/l. The cost reduction of TGE-produced proteins, due to significant decrease in pDNA amount requirement for transfections, will certainly facilitate the development and implementation of large scale TGE as a feasible method for TGE-based protein production.

EPFL2013

Development of a new reporter system for the detection of protein-protein interactions in living cells

Petra Tafelmeyer

Split-protein sensors have become an important tool for the analysis of protein-protein interactions in living cells. In general, two interacting proteins are expressed as fusion proteins with a pair of inactive fragments of a reporter enzyme. Interaction-induced reassembly of the two fragments then results in a functional enzyme and a detectable phenotypic readout. Despite the constantly expanding repertoire of methods, new split-protein sensors that could detect and screen for protein-protein interactions both in the cytosol and in the membrane would be very useful. In a first attempt to create new split-protein sensors, cytochrome c peroxidase (CCP) from the yeast Saccharomyces cerevisiae was rationally dissected into two fragments, which were fused to two interacting proteins. Activity of reassembled peroxidase might be visually detected using a simple colony screen [1]. However, this approach failed due to the insolubility of the chosen fragments. A random approach based on circular permutation originally developed by Graf and Schachmann was therefore adapted to isolate suitable fragmentation sites [2]. Unfortunately, only split-proteins expressing quasi wild-type protein were isolated, resulting from cuts close to the N or the C terminus of CCP. Two reasons can account for this: (i) the fragile active site environment of CCP could considerably hamper the fragmentation of the enzyme; (ii) the method itself favors the isolation of quasi wild-type proteins. The combinatorial method for the generation of new split-protein sensors was therefore further modified to circumvent the isolation of quasi wild-type proteins and successfully applied to the (β/α)8-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. In addition, split-Trp can be used as reporter for the detection of small molecule-protein interactions. This powerful selection thus complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening. Furthermore, the combinatorial approach should be able to generate split-protein sensors of almost any protein, thereby yielding tailor-made sensors for different applications.

EPFL2004

Directed Evolution of O6-Alkylguanine-DNA Alkyltransferase for Efficient Labeling of Fusion Proteins with Small Molecules In Vivo

Thomas Gronemeyer, Kai Johnsson, Alexandre Juillerat, Antje Keppler, Horst Pick, Horst Vogel

We report here the generation of mutants of the human O6-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter mols. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O6-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O6-benzylguanine derivs., the selected mutant proteins allow for a highly efficient covalent labeling of hAGT fusion proteins in vivo and in vitro with small mols. and therefore should become important tools for studying protein function in living cells. In addn. to various applications in proteomics, the selected mutants also yield insight into the interaction of the DNA repair protein hAGT with its inhibitor O6-benzylguanine. [on SciFinder (R)]