Alexey N. Fedorov

Folding on the ribosome of Escherichia coli tryptophan synthase β subunit nascent chains probed with a conformation-dependent monoclonal antibody

Experimental analysis of protein folding during protein synthesis on the ribosome is rendered very difficult by the low concentration of nascent polypeptides and the heterogeneity of the translation mixture. In this study, an original approach is developed for analysing nascent polypeptide structures still carried by the ribosome. Folding on the ribosome of nascent chains of the beta subunit of Escherichia coli tryptophan synthase was investigated using a monoclonal antibody (mAb 19) recognizing a conformation-dependent antigenic determinant. Upon synthesis of beta subunits in an E. coli coupled transcription-translation system, it is shown that ribosome-bound nascent polypeptides can react with the monoclonal antibody provided their size is above 11.5 kDa, which is smaller than that of both the N-terminal proteolytic and crystallographic domains (29 and 21 kDa, respectively). The gene fragments coding only for the 11.5 kDa polypeptide, with and without stop codon at the end of the corresponding mRNAs, were constructed and expressed in a cell-free wheat germ translation system. It is shown that antibody 19 reacts with this polypeptide either bound to the ribosome or free in solution. That the 11.5 kDa polypeptide acquires a condensed structure is shown by gel filtration in native conditions and by urea gradient gel electrophoresis. Moreover, it is demonstrated that this condensed structure resembles that of native beta 2 in the vicinity of the epitope for antibody 19. Indeed, the affinity of antibody 19 for the 11.5 kDa fragment, either free or bound to the ribosome, was measured (6 x 10(8) M-1) and shown to be close to that for native beta 2. It is therefore proposed that the polypeptide chain may start to fold during its biosynthesis and that, even before the appearance of an entire domain, a folded intermediate is formed that already exhibits some local structural features of the native state and of an immunoreactive intermediate previously detected during the in vitro refolding of denatured complete beta chains.

De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure. Probing the structure at the nanogram level.

The de novo polypeptide named albebetin was designed to form the tertiary fold that has not yet been observed in natural proteins. The design was based on the molecular theory of protein structures. The gene coding for this polypeptide was chemically synthesized. For the initial characterization of a protein structure, a new approach has been developed that uses only nanogram amounts of a polypeptide without its previous purification. This approach includes the biosynthesis of radiolabeled protein in a cell-free translation system with subsequent analysis of its compactness and structure by size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis. According to all tests used, albebetin has a compact stable structure.

Influence of exon duplication on intron and exon phase distribution.

Abstract. Nonrandomness in the intron and exon phase distributions in a sample of 305 human genes has been found and analyzed. It was shown that exon duplications had a significant effect on the exon phase nonrandomness. All of the nonrandomness is probably due to both the processes of exon duplication and shuffling. A quantitative estimation of exon duplications in the human genome and their influence on the intron and exon phase distributions has been analyzed. According to our estimation, the proportion of duplicated exons in the human genome constitutes at least 6% of the total. Generalizing the particular case of exon duplication to the more common event of exon shuffling, we modeled and analyzed the influence of exon shuffling on intron phase distribution.

Variable and Invariable DNA Repeat Characters Revealed by Taxonprint Approach are Useful for Molecular Systematics

Abstract. A specially optimized restriction analysis of highly repetitive DNA elements, called DNA taxonprint, was applied for phylogenetic study of primates and lizards. It was shown that electrophoretic bands of DNA repeats revealed by the taxonprint technique have valuable properties for molecular systematics. Approximately half of taxonprint bands (TB) are invariable and do not disappear from the genomes during evolution or change spontaneously. Presumably these invariable bands are restriction fragments of dispersed DNA repeats. Another group represents variable taxonprint bands that differ even between closely related species. These variable bands are probably represented by tandem DNA repeats and could be used as species-specific markers. It was shown that taxonprint bands are independent characters since the appearance of a new taxonprint band does not change the previous band pattern. Phylogenetic reconstruction carried out on taxonprint data demonstrated that this approach could be of general utility for molecular systematics and species identification.

Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography.

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.

High-efficient expression, refolding and purification of functional recombinant C-terminal fragment of human alpha-fetoprotein.

Human alpha-fetoprotein (hAFP) is an oncofetal protein which is a common cancer marker. Conjugates of native hAFP with different cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The hAFP interacts with its receptor (AFPR) on the surface of cancer cells via its C-terminal domain. The aim of this work was to develop a highly efficient expression system in Escherichia coli and efficient refolding procedure for the recombinant C-terminal fragment of hAFP (rAFP-Cterm) and to characterize its functional properties. C-terminal fragment of hAFP (rAFP-Cterm) comprising amino acids from 404 to 609 was expressed in E. coli BL21 (DE3) strain with high yield. High efficient purification and refolding procedures were developed giving yield of refolded protein about 80% with purity about 95%. The refolded rAFP-Cterm bound specifically with cancer cells carrying AFPR and was accumulated by them with the same efficiency as native hAFP. This rAFP-Cterm can be used as a vehicle for the targeted delivery of drugs to cancer cells.

Study of rubella candidate vaccine based on a structurally modified plant virus

Abstract A novel rubella candidate vaccine based on a structurally modified plant virus – spherical particles (SPs) – was developed. SPs generated by the thermal remodelling of the tobacco mosaic virus are promising platforms for the development of vaccines. SPs combine unique properties: biosafety, stability, high immunogenicity and the effective adsorption of antigens. We assembled in vitro and characterised complexes (candidate vaccine) based on SPs and the rubella virus recombinant antigen. The candidate vaccine induced a strong humoral immune response against rubella. The IgG isotypes ratio indicated the predominance of IgG1 which plays a key role in immunity to natural rubella infection. The immune response was generally directed against the rubella antigen within the complexes. We suggest that SPs can act as a platform (depot) for the rubella antigen, enhancing specific immune response. Our results demonstrate that SPs‐antigen complexes can be an effective and safe candidate vaccine against rubella. Graphical abstract Figure. No caption available. HighlightsIn vitro assembly and characterisation of a rubella candidate vaccine based on modified plant virus (spherical particles).Candidate vaccine induces efficient humoral immune response against rubella.Acute and chronic toxicity studies demonstrate biosafety of candidate vaccine.Candidate vaccine demonstrated good results and can be considered as a promising safe tool against the rubella virus.

In vitro gene expression for the localization of antigenic determinants: application to the E. coli tryptophan synthase β2 subunit

Gene expression of the beta subunit of E. coli tryptophan synthase in an E. coli cell-free transcription-translation system proceeds by pauses and produces a discrete but quite continuous pattern of nascent chains starting from the N terminus and ranging in size up to the 44 kDa end product corresponding to the completed beta chains. Using specific immunoadsorption of [35S]Met radiolabelled nascent chains by different monoclonal antibodies directed against the beta 2 subunit of E. coli tryptophan synthase, the size of the smallest N-terminal fragment reacting with each antibody has been determined by SDS electrophoretic analysis of the immunoadsorbed polypeptides. The immunoadsorption assay is performed in solution under conditions avoiding the usual drawbacks of solid phase immunoassay. This approach, in combination with the results obtained with a DNA fragment library permitted us to localize the antigenic determinants recognized by the monoclonal antibodies. The proposed method could help to localize rapidly the C-terminal boundary of an epitope, before starting systematic and precise mapping by other approaches. Moreover, the method described may be of general interest for the rapid production of a large set of C-terminal truncated polypeptides for studies of antigen-antibody recognition.

A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.

We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification.

Assessment of structurally modified plant virus as a novel adjuvant in toxicity studies

ABSTRACT Spherical particles (SPs) generated by thermally denatured tobacco mosaic virus (TMV) coat protein can act as an adjuvant, as they are able to enhance the magnitude and longevity of immune responses to different antigens. Here, the toxicity of TMV SPs was assessed prior to it being offered as a universal safe adjuvant for the development of vaccine candidates. The evaluation included nonclinical studies of a local tolerance following the single administration of TMV SPs, and of the local and systemic effects following repeated administrations of TMV SPs. These were conducted in mice, rats and rabbits. General health status, haematology and blood chemistry parameters were monitored on a regular basis. Also, reproductive and development toxicity were studied. No significant signs of toxicity were detected following single or repeated administrations of the adjuvant (TMV SPs). The absence of toxicological effects following the injection of TMV SPs is promising for the further development of recombinant vaccine candidates with TMV SPs as an adjuvant. HIGHLIGHTSThe toxic effects of TMV SPs (structurally modified helical plant virus) were studied.Minor reversible deviations in histology and blood tests are observed at high doses of TMV SPs.Immunotoxicity, reproductive and developmental toxicity of TMV SPs are not detected.TMV SPs are the promising and safe adjuvant for new vaccine candidates development.