Sergey Mureev

Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae.

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae. DOI: http://dx.doi.org/10.7554/eLife.01434.001

Leishmania cell-free protein expression system

Cell-free protein expression is an important tool for a rapid production, engineering and labeling of recombinant proteins. However the complex protocols for preparation of eukaryotic cell-free protein expression systems result in high manufacturing costs and limit their utility. Recently we reported a novel cell-free expression system based on the lysate of a fermentable protozoan Leishmania tarentolae. Herein we describe a protocol for high throughput protein expression using Leishmania cell-free lysate. The protocol combines PCR-based synthesis and engineering of translation templates with a combined transcription-translation system. The protocol is adapted to multiwell plate format and allows translation of large protein libraries. In the presented example we translate in vitro and isolate a nearly complete complement of mammalian Rab GTPases. Further applications and developments of the system are discussed.

Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System

The adaptation of organisms to a parasitic life style is often accompanied by the emergence of novel biochemical pathways absent in free-living organisms. As a result, the genomes of specialized parasitic organisms often code for a large number (>50%) of proteins with no detectable homology or predictable function. Although understanding the biochemical properties of these proteins and their roles in parasite biogenesis is the next challenge of molecular parasitology, analysis tools developed for free-living organisms are often inadequate for this purpose. Here we attempt to solve some of these problems by developing a methodology for the rapid production of expressed proteomes in cell-free systems based on parasitic organisms. To do so we take advantage of Species Independent Translational Sequences (SITS), which can efficiently mediate translation initiation in any organism. Using these sequences we developed a single-tube in vitro translation system based on the parasitic protozoan Leishmania tarentolae. We demonstrate that the system can be primed directly with SITS containing templates constructed by overlap extension PCR. To test the systems we simultaneously amplified 31 of L. tarentolaes putative translation initiation factors and phosphatases directly from the genomic DNA and subjected them to expression, purification and activity analysis. All of the amplified products produced soluble recombinant proteins, and putative phosphatases could be purified to at least 50% purity in one step. We further compared the ability of L. tarentolae and E. coli based cell-free systems to express a set of mammalian, L. tarentolae and Plasmodium falciparum Rab GTPases in functional form. We demonstrate that the L. tarentolae cell-free system consistently produced higher quality proteins than E. coli-based system. The differences were particularly pronounced in the case of open reading frames derived from P. falciparum. The implications of these developments are discussed.

Rapid Mapping of Interactions between Human SNX-BAR Proteins Measured In Vitro by AlphaScreen and Single-molecule Spectroscopy

Protein dimerization and oligomerization is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to transfer information in signaling, transcriptional, and membrane trafficking events. Here we show that a combination of cell-free protein expression, a proximity based interaction assay (AlphaScreen), and single-molecule fluorescence allow rapid mapping of homo- and hetero-oligomerization of proteins. We have applied this approach to the family of BAR domain-containing sorting nexin (SNX-BAR) proteins, which are essential regulators of membrane trafficking and remodeling in all eukaryotes. Dimerization of BAR domains is essential for creating a concave structure capable of sensing and inducing membrane curvature. We have systematically mapped 144 pairwise interactions between the human SNX-BAR proteins and generated an interaction matrix of preferred dimerization partners for each family member. We find that while nine SNX-BAR proteins are able to form homo-dimers, several including the retromer-associated SNX1, SNX2, and SNX5 require heteromeric interactions for dimerization. SNX2, SNX4, SNX6, and SNX8 show a promiscuous ability to bind other SNX-BAR proteins and we also observe a novel interaction with the SNX3 protein which lacks the BAR domain structure.

Performance Benchmarking of Four Cell-Free Protein Expression Systems

Over the last half century, a range of cell‐free protein expression systems based on pro‐ and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species‐independent translation initiation sequence to express and characterize 87 N‐terminally GFP‐tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania‐based (LTE) cell‐free systems. Using a combination of single‐molecule fluorescence spectroscopy, SDS‐PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full‐length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species—particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE‐produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa‐ and LTE‐produced proteins enable their analysis without purification and make them suitable for analysis of multi‐domain eukaryotic proteins. Biotechnol. Bioeng. 2016;113: 292–300.

Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems

Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era.

Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis

Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.

Combining Sense and Nonsense Codon Reassignment for Site-Selective Protein Modification with Unnatural Amino Acids

Incorporation of unnatural amino acids (uAAs) via codon reassignment is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. However, the site-selective incorporation of multiple uAAs into polypeptides is hampered by the limited number of reassignable nonsense codons. This challenge is addressed in the current work by developing Escherichia coli in vitro translation system depleted of specific endogenous tRNAs. The translational activity in this system is dependent on the addition of synthetic tRNAs for the chosen sense codon. This allows site-selective uAA incorporation via addition of tRNAs pre- or cotranslationally charged with uAA. We demonstrate the utility of this system by incorporating the BODIPY fluorophore into the unique AGG codon of the calmodulin(CaM) open reading frame using in vitro precharged BODIPY-tRNACysCCU. The deacylated tRNACysCCU is a poor substrate for Cysteinyl-tRNA synthetase, which ensures low background incorporation of Cys into the chosen codon. Simultaneously, p-azidophenylalanine mediated amber-codon suppression and its post-translational conjugation to tetramethylrhodamine dibenzocyclooctyne (TAMRA-DIBO) were performed on the same polypeptide. This simple and robust approach takes advantage of the compatibility of BODIPY fluorophore with the translational machinery and thus requires only one post-translational derivatization step to introduce two fluorescent labels. Using this approach, we obtained CaM nearly homogeneously labeled with two FRET-forming fluorophores. Single molecule FRET analysis revealed dramatic changes in the conformation of the CaM probe upon its exposure to Ca2+ or a chelating agent. The presented approach is applicable to other sense codons and can be directly transferred to eukaryotic cell-free systems.

Subunit organisation of in vitro reconstituted HOPS and CORVET multisubunit membrane tethering complexes.

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data.

Oligonucleotide-mediated tRNA sequestration enables one-pot sense codon reassignment in vitro

Abstract Sense codon reassignment to unnatural amino acids (uAAs) represents a powerful approach for introducing novel properties into polypeptides. The main obstacle to this approach is competition between the native isoacceptor tRNA(s) and orthogonal tRNA(s) for the reassigned codon. While several chromatographic and enzymatic procedures for selective deactivation of tRNA isoacceptors in cell-free translation systems exist, they are complex and not scalable. We designed a set of tRNA antisense oligonucleotides composed of either deoxy-, ribo- or 2′-O-methyl ribonucleotides and tested their ability to efficiently complex tRNAs of choice. Methylated oligonucleotides targeting sequence between the anticodon and variable loop of tRNASerGCU displayed subnanomolar binding affinity with slow dissociation kinetics. Such oligonucleotides efficiently and selectively sequestered native tRNASerGCU directly in translation-competent Escherichia coli S30 lysate, thereby, abrogating its translational activity and liberating the AGU/AGC codons. Expression of eGFP protein from the template harboring a single reassignable AGU codon in tRNASerGCU-depleted E. coli lysate allowed its homogeneous modification with n-propargyl-l-lysine or p-azido-l-phenylalanine. The strategy developed here is generic, as demonstrated by sequestration of tRNAArgCCU isoacceptor in E. coli translation system. Furthermore, this method is likely to be species-independent and was successfully applied to the eukaryotic Leishmania tarentolae in vitro translation system. This approach represents a new direction in genetic code reassignment with numerous practical applications.