Leonid M. Vinokurov

A genetically encoded sensor for H2O2 with expanded dynamic range

Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H(2)O(2)] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H(2)O(2) due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicators dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H(2)O(2)] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.

Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells

Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.

Method for real-time monitoring of protein degradation at the single cell level

Protein degradation is important in practically all aspects of cellular physi-ology (1). Together with transcription and translation, proteolysis ensures maintenance and rapid regulation of individual protein concentration in living cells. The half-life for different proteins varies between a few minutes and days. Moreover, decay rate for many proteins change drastically throughout the cell cycle or in response to external stimuli.Two methods are commonly used to determine a protein’s half-life, namely radioactive pulse-chase analysis and cycloheximide chase (2). Pulse-chase analysis provides minimal distortion of normal cell physiology. The main disadvantages of this method are its laboriousness and necessity for radio-labeling. In contrast to pulse-chase analysis, cycloheximide chase strongly affects cellular metabolism that should be considered a serious disadvantage for this approach. Importantly, both methods do not allow real-time measurements at the single cell level.The use of green fluorescent protein (GFP) provided an opportunity to apply fluorescence microscopy and flow cytometry to protein degradation analysis (3–6). However, to extract information regarding protein degra-dation using GFP, one should block the synthesis of new GFP molecules (e.g., by cycloheximide) (3,4). Even in presence of cycloheximide, residual GFP chromophore maturation affects estimation of degradation rate, especially when maturation and degra-dation half-times are comparable.Since 2002, a number of so-called photoactivatable fluorescent proteins (PAFPs) have been developed (7). Most commonly, local PAFP activation is used to visualize protein movement within cells. Here we propose to use PAFP activation within a whole cell to monitor protein degradation (Figure 1). Indeed, while steady-state fluorescence intensity of an FP-tagged protein depends on both synthesis and degradation rates, photo-activation creates a fluorescent signal that depends only on protein degra-dation (fluorescence bleaching during observation should be taken into account and made allowance for). Thus, time-lapse imaging of activated PAFP allows quantification of the tagged protein degradation process. Also, this protocol can be potentially adapted for PAFP photoactivation in large cell culture samples and their further analysis by flow cytometry or microplate readers.To demonstrate the usability of the method proposed, we used the green-to-red photoconvertible fluorescent protein Dendra2 (Evrogen, Moscow, Russia), which is a commercially available improved version of Dendra (8). In the dark, Dendra2 matures up to the green fluorescent state with excitation-emission maxima at 490 and 507 nm, respectively. Upon maturation, it can be converted into a red-emitting protein (excitation-emission at 553/573 nm) by irradiation with violet (e.g., 405 nm) or blue (e.g., 488 nm) light. Due to its monomeric state, Dendra2 can be safely used for protein labeling. In contrast to other PAFPs, Dendra2 can be activated with blue light, which is less damaging compared with ultraviolet (UV) or violet light. Using a well-established assay based on dithionite reduction of chromo-phore of urea-denatured fluorescent protein followed by dilution of the sample and maturation of the fluorescent protein starting from the native polypeptide (9), we measured Dendra2 maturation half-time as 90 min at 37°C.Similar to GFP, Dendra2 was found to be a long-lived protein. In HeLa and HEK 293 cells, we observed no decrease in Dendra2 green fluorescence for several hours after the addition of cycloheximide (not shown). To photoconvert Dendra2 throughout the experiments, we applied 10–20 s of irradiation with blue light from a 100 W Hg-lamp using the GFP filter set. As a result, the green signal decreased while clearly detectable red fluores-cence appeared. Then, we monitored red fluorescence at 37°C in the confocal mode (Leica DMIRE2 TCS SP2 micro-scope; Leica Microsystems GmbH, Wetzlar, Germany) using the 543-nm laser line. Practically no decay of red fluorescence after Dendra2 photocon-version was observed (Figure 2A), demonstrating very high stability of this protein in living cells. Next we tested the influence of peptides or proteins known to determine fast degra-

EGFP as a fusion partner for the expression and organic extraction of small polypeptides

Green fluorescent protein (GFP) is widely used as an excellent reporter module of the fusion proteins. The unique structure of GFP allows isolation of the active fluorescent protein directly from the crude cellular sources by extraction with organic solvents. We demonstrated the stable expression of four short polypeptides fused to GFP in Escherichia coli cells, including antimicrobial cationic peptides, which normally kill bacteria. EGFP module protected fusion partners from the intracellular degradation and allowed the purification of the chimerical proteins by organic extraction. The nature of the polypeptide fused to GFP, as opposed to the order of GFP and the polypeptide modules in the fusion protein, influenced the efficiency of the described purification technique.

The primary structure of the 5 S RNA binding protein L25 from Escherichia coli ribosomes

Protein L25 was isolated from E. coli MRE-600 70 S ribosomes as described earlier [l] with a yield of about 80 mg from 10 g of the total 70 S ribosome protein. The peptides derived by cleavage of the protein molecule with trypsin, with cyanogen bromide, of the modified protein with trypsin only at the arginine residues [3], as well as by cleavage of cyanogen bromide fragments with bromosuccinimide (at the tyrosine residues [4] ) were separated by gelfiltration on Sephadex G-50, chromatography on Aminex-A-5 resin, paper chromatography and paper high-voltage electrophoresis. The amino acid composition was determined on the amino acid analyzer BC-201 (Bio-Cal, BRD). The amino acid sequence of the isolated peptides was determined by Edman’s phenylisothiocyanate method, the dansyl Edman method [5] and by automated Edman degradation on a sequenser (model 89OC, Beckman, USA) using fast and slow peptide programs. The amino acid sequence of peptides T-10 and BS-2 was determined by automated degradation with a modification of lysine residues according to Braunitzer [6]. PTH-derivatives of amino acids were identified by gas-liquid and thinlayer chromatography, the DNS-derivatives by twodimensional thin-layer chromatography on silica gel [7,8]. The C-terminal amino acid residues of protein and peptides were determined by carboxypeptidases A and B [9].

The primary structure of elongation factor from Escherichia coli: A complete amino acid sequence

Elongation factor G (EF-G) is a large protein consisting of one polypeptide chain with MI 80 000. Studies of this protein by traditional methods used to determine the primary structure did not seem the most rational. We chose the method of limited proteolysis as the first stage of studies permitting, in many cases, cleavage of the protein molecule into a small number of large fragments relatively stable against further effects of protease. On mild tryptic hydrolysis the G-factor is split into 5 fragments [ 1, cf. 2,3], 4 of which (T,-T,) form the complete protein poly peptide chain (for fragment nomenclature and arrangement in the protein chain see fig.1). Thus, the studies of the primary structure of the elongation factor G led to determination of the structure of these 4 fragments and a search for the overlapping peptides for reconstitution of the entire polypeptide chain. Studies of the structure of fragments TS-T, were relatively simple due to their comparatively low molecular mass. The molecular mass of the N-terminal fragment T6 is 6500, that of the following fragment T,, 7500, and that of the C-terminal fragment Ts, 25 000. Traditional chemical and enzymatic methods of polypeptide chain cleavage permitted us earlier to determine the complete primary structures of fragments Tb [4], T, [5,6] and Ts [7] involving a total of >350 amino acid residues. To elucidate the complete structure of elongation factor G it was necessary to determine the ammo acid sequence of the largest tryptic fragment T4 representing the middle part of the protein molecule and to obtain the peptides joining all the fragments of limited trypsinolysis into a 1 polypeptide chain. To this end, the products of protein cleavage by cyanogen bromide and those of limited acidic hydrolysis at the Asp-Pro bond were studied. This paper is devoted to the results of these studies which permitted us to join all the fragments of limited trypsinolysis into one polypeptide chain and to present the complete amirmacid sequence of the elongation factor G.

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

A Catecholic Siderophore Produced by the Thermoresistant Bacilluslicheniformis VK21 Strain

Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for the producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe3+, in response to addition of manganese(II) salts. The growth of the producer on a minimal medium containing MnSO4 under the conditions of iron deficiency is accompanied by the accumulation of a catecholic product, the content of which reaches maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl3, the amount of the catecholic product in the medium considerably decreases. The siderophore, called SVK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA. The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm. According to the amino acid analysis and NMR spectrometry, the metabolite SVK21 is 2,3-dihydroxybenzoyl-glycyl-threonine.

Lifetime imaging of FRET between red fluorescent proteins

Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties.

Universal and rapid method for purification of GFP-like proteins by the ethanol extraction

GFP-like fluorescent proteins (FPs) are crucial in biological and biomedical studies. The majority of FP purification techniques either include multiple time-consuming chromatography steps with a low yield of the desired product or require prior protein modification (addition of special tags). In the present work, we propose an alternative ethanol extraction-based technique previously used for GFP purification and then modified for diverse FPs originated from different sources. The following recombinant FPs were expressed using Escherichia coli M15 (pREP4) strain as a host transformed with pQE30 plasmid bearing one of the target FP genes: TagCFP, TagGFP, TagYFP, TagRFP, TurboGFP, TurboRFP, Dendra2, TurboFP602 and KillerRed. Despite their diversity, all tested recombinant FPs were successfully purified and yielded a highly homogeneous product. The method is easily scalable for purification of any amount of protein and requires no expensive reagents and equipment.