Caterina Holz

A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae

Methods have been developed aimed at applying at high-throughput technology for expression of cloned cDNAs in yeast. Yeast is a eukaryotic host, which produces soluble recombinant proteins and is capable of introducing post-translational modifications of protein. It is, thus, an appropriate expression system both for the routine expression of various cDNAs or protein domains and for the expression of proteins, which are not correctly expressed in Escherichia coli. Here, we describe a standard system in Saccharomyces cerevisiae, based on a vector for intracellular protein expression, where the gene products are fused to specific peptide sequences (tags). These epitope tags, the N-terminal His(6) tag and the C-terminal StrepII tag, allow subsequent immunological identification and purification of the gene products by a two-step affinity chromatography. This method of dual-tagged recombinant protein purification eliminates contamination by degraded protein products. A miniaturization of the procedures for cloning, expression, and detection was performed to allow all steps to be carried out in 96-well microtiter plates. The system is, thus, suitable for automation. We were able to analyze the simultaneous protein expression of a large number of cDNA clones due to the highly parallel approach of protein production and purification. The microtiter plate technology format was extended to quantitative analysis. An ELISA-based assay was developed that detects StrepII-tagged proteins. The application of this high-throughput expression system for protein production will be a useful tool for functional and structural analyses of novel genes, identified by the Human Genome Project and other large-scale sequencing projects.

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis‐Golgi membrane. The crystal structure of human BET3 has been determined to 1.55‐Å resolution. BET3 adopts an α/β‐plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane‐associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein–protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.

Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin

As a key regulator of mitosis, the Ser/Thr protein polo-like kinase-1 (Plk-1) is a well validated drug target in cancer therapy. In order to enable structure-guided drug design, determination of the crystal structure of the kinase domain of Plk-1 was attempted. Using a multi-parallel cloning and expression approach, a set of length variants were identified which could be expressed in large amounts from insect cells and which could be purified to high purity. However, all attempts to crystallize these constructs failed. Crystals were ultimately obtained by generating designed ankyrin-repeat proteins (DARPins) selective for Plk-1 and using them for cocrystallization. Here, the first crystal structure of the kinase domain of wild-type apo Plk-1, in complex with DARPin 3H10, is presented, underlining the power of selective DARPins as crystallization tools. The structure was refined to 2.3 A resolution and shows the active conformation of Plk-1. It broadens the basis for modelling and cocrystallization studies for drug design. The binding epitope of 3H10 is rich in arginine, glutamine and lysine residues, suggesting that the DARPin enabled crystallization by masking a surface patch which is unfavourable for crystal contact formation. Based on the packing observed in the crystal, a truncated DARPin variant was designed which showed improved binding characteristics.

Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics

We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.

Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale

Structural genomics requires the application of a standardised process for overexpression of soluble proteins that allows high-throughput purification and analysis of protein products. We have developed a highly parallel approach to protein expression, including the simultaneous expression screening of a large number of cDNA clones in an appropriate vector system and the use of a protease-deficient host strain. A set of 221 human genes coding for proteins of various sizes with unknown structures was selected to evaluate the system. We transferred the cDNAs from an E. coli vector to the yeast expression vector by recombinational cloning, avoiding time-consuming recloning steps and the use of restriction enzymes in the cloning process. The subcloning yield was 95%, provided that a PCR fragment of the correct size could be obtained. Sixty percent of these proteins were expressed as soluble products at detectable levels and 48% were successfully purified under native conditions using the His6 tag fusion.The advantages of the developed yeast-based expression system are the ease of manipulation and cultivation of S. cerevisiae in the same way as with prokaryotic hosts and the ability to introduce post-translational modifications of proteins if required, thus being an attractive system for heterologous expression of mammalian proteins. The expression clones selected in this screening process are passed on to the fermentation process in order to provide milligram amounts of proteins for structure analysis within the ‘Berlin Protein Structure Factory’. All data generated is stored in a relational database and is available on our website(http://www.proteinstrukturfabrik.de).

Enhanced glutathione production by evolutionary engineering of Saccharomyces cerevisiae strains

Glutathione is an important natural tripeptide mainly used because of its antioxidative properties. Commercial glutathione is microbially synthesized by yeasts and the growing demand requires the development of new production strains. An adaptive laboratory evolution strategy using acrolein as a selection agent was employed to obtain strains with an enhanced glutathione accumulation phenotype accompanied by an acrolein resistance phenotype. Two particularly interesting isolates were obtained: one with a high volumetric productivity for glutathione reaching 8.3 mgglutathione/L h, which is twice as high as the volumetric productivity of its parental strain. This strain reached an elevated intracellular glutathione content of 3.9%. A second isolate with an even higher acrolein tolerance exhibited a lower volumetric productivity of 5.8 mgglutathione/L h due to a growth phenotype. However, this evolved strain accumulated glutathione in 3.3‐fold higher concentration compared to its parental strain and reached a particularly high glutathione content of almost 6%. The presented results demonstrate that acrolein is a powerful selection agent to obtain high glutathione accumulation strains in an adaptive laboratory evolution experiment.

Significant Reduction in Helicobacter pylori Load in Humans with Non-viable Lactobacillus reuteri DSM17648: A Pilot Study.

Abstract Reducing the amount of Helicobacter pylori in the stomach by selective bacterial–bacterial cell interaction was sought as an effective and novel method for combating the stomach pathogen. Lactobacillus reuteri DSM17648 was identified as a highly specific binding antagonist to H. pylori among more than 700 wild-type strains of Lactobacillus species. Applying a stringent screening procedure, the strain DSM17648 was identified as selective binder to H. pylori cells under in vivo gastric conditions. The strain DSM17648 co-aggregates the pathogen in vivo and in vitro. The specific co-aggregation occurs between Lact. reuteri DSM17648 and different H. pylori strains and serotypes, as well as H. heilmannii, but not with Campylobacter jejuni or other commensal oral and intestinal bacteria. Lact. reuteri DSM17648 was shown in a proof-of-concept single-blinded, randomized, placebo-controlled pilot study to significantly reduce the load of H. pylori in healthy yet infected adults. Reducing the amount of H. pylori in the stomach by selective bacterial–bacterial cell interaction might be an effective and novel method for combating the stomach pathogen. Lact. reuteri DSM17648 might prove useful as an adhesion blocker in antibiotic-free H. pylori therapies.

High-Throughput Expression in Microplate Format in Saccharomyces cerevisiae

We have developed a high-throughput technology that allows parallel expression, purification, and analysis of large numbers of cloned cDNAs in the yeast Saccharomyces cerevisiae. The technology is based on a vector for intracellular protein expression under control of the inducible CUP1 promoter, where the gene products are fused to specific peptide sequences. These N-terminal and C-terminal epitope tags allow the immunological identification and purification of the gene products independent of the protein produced. By introducing the method of recombinational cloning we avoid time-consuming re-cloning steps and enable the easy switching between different expression vectors and host systems.

Ribosomally synthesized antimicrobial peptides in prokaryotic and eukaryotic organisms

Abstract Ribosomally synthesized antimicrobial peptides are found both in prokaryotes and eukaryotes. They possess a low molecular weight and are characterized by compact structures, thermal stability and potent antimicrobial activity. Their typical amphipathic character is responsible for their detergent‐like mode of action. Since these peptides are directly gene‐encoded, it is possible to modify their structure by mutagenesis to produce structure analogues with an expanded spectrum of antimicrobial activity. Furthermore, the production of these peptides in heterologous expression systems could enable yield improvement to make them accessible for industrial application and to reduce the processing costs by using optimized isolation procedures. They might be useful as effective natural food preservatives because of their chemical and physical properties.

Impact of glutathione metabolism on zinc homeostasis in Saccharomyces cerevisiae

Zinc is a crucial mineral for all organisms as it is an essential cofactor for the proper function of a plethora of proteins and depletion of zinc causes oxidative stress. Glutathione is the major redox buffering agent in the cell and therefore important for mitigation of the adverse effects of oxidative stress. In mammalian cells, zinc deficiency is accompanied by a glutathione depletion. In the yeast Saccharomyces cerevisiae, the opposite effect is observed: under low zinc conditions, an elevated glutathione concentration is found. The main regulator to overcome zinc deficiency is Zap1p. However, we show that Zap1p is not involved in this glutathione accumulation phenotype. Furthermore, we found that in glutathione-accumulating strains also the metal ion-binding phytochelatin-2, which is an oligomer of glutathione, is accumulated. This increased phytochelatin concentration correlates with a lower free zinc level in the vacuole. These results suggest that phytochelatin is important for zinc buffering in S. cerevisiae and thus explains how zinc homeostasis is connected with glutathione metabolism.