MS2 phage-like particles (MS2 PLP) are artificially constructed pseudo-viral particles derived from bacteriophage MS2. They are able to carry a specific single stranded RNA (ssRNA) sequence of choice inside their capsid, thus protecting it against the effects of ubiquitous nucleases. Such particles are able to mimic ssRNA viruses and, thus, may serve as the process control for molecular detection and quantification of such agents in several kinds of matrices, vaccines and vaccine candidates, drug delivery systems, and systems for the display of immunologically active peptides or nanomachines. Currently, there are several different in vivo plasmid-driven packaging systems for production of MS2 PLP. In order to combine all the advantages of the available systems and to upgrade and simplify the production and purification of MS2 PLP, a one-plasmid double-expression His-tag system was designed. The described system utilizes a unique fusion insertional mutation enabling purification of particles using His-tag affinity. Using this new production system, highly pure MS2 PLP can be quickly produced and purified by a fast performance liquid chromatography (FPLC) approach. The system can be easily adapted to produce other MS2 PLP with different properties.
- MeSH
- Levivirus * chemie genetika metabolismus MeSH
- plazmidy * genetika metabolismus MeSH
- rekombinantní fúzní proteiny * biosyntéza chemie genetika izolace a purifikace MeSH
- virion * chemie genetika izolace a purifikace metabolismus MeSH
- virové plášťové proteiny * biosyntéza chemie genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Arabidopsisthaliana pathogenesis-related 1 (PR1) is an important defense protein, so far it has only been detected in extracellular space and its subcellular sorting and transport remain unexplained. Using a green fluorescent protein (GFP) tagged full length, as well as a C-terminus truncated version of PR1, we observed that when expressed ectopically in Nicotiana benthamiana leaves, PR1 co-localizes only partially with Golgi markers, and much more prominently with the late endosome (LE)/multivesicular body (MVB) FYVE marker. The C-truncated version PR1ΔC predominantly localized to the endoplasmic reticulum (ER). The same localizations were found for stable Arabidopsis transformants with expression of PR1 and PR1ΔC driven by the native promoter. We conclude that the A. thaliana PR1 (AtPR1) undergoes an unconventional secretion pathway, starting from the C-terminus-dependent sorting from the ER, and utilizing further transportation via phosphatidyl-inositol-3-phosphate (PI(3)P) positive LE/MVB-like vesicles. The homology model of the PR1 structure shows that the cluster of positively charged amino acid residues (arginines 60, 67, 137, and lysine 135) could indeed interact with negatively charged phospholipids of cellular membranes. It remains to be resolved whether Golgi and LE/MVB localization reflects an alternative sorting or trafficking succession, and what the role of lipid interactions in it will be.
- MeSH
- Arabidopsis metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- endozomy metabolismus MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- Golgiho aparát metabolismus MeSH
- konfokální mikroskopie MeSH
- listy rostlin metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- proteiny huseníčku genetika metabolismus MeSH
- rekombinantní fúzní proteiny biosyntéza genetika MeSH
- tabák metabolismus MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.
- MeSH
- aktivace enzymů MeSH
- Baculoviridae genetika metabolismus MeSH
- genetické vektory genetika metabolismus MeSH
- HEK293 buňky MeSH
- hmyz genetika metabolismus MeSH
- imunoglobulin A genetika metabolismus MeSH
- imunoglobuliny - kappa-řetězce chemie genetika MeSH
- klonování DNA MeSH
- kultivační média metabolismus MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- N-acetylgalaktosaminyltransferasy biosyntéza genetika izolace a purifikace MeSH
- plazmidy genetika metabolismus MeSH
- proteiny - lokalizační signály MeSH
- rekombinantní fúzní proteiny biosyntéza genetika izolace a purifikace MeSH
- rozpustnost MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů MeSH
- tandemová hmotnostní spektrometrie MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- srovnávací studie MeSH
Proteins MPT63 and MPT83 which are common for both Mycobacterium tuberculosis and Mycobacterium bovis, due to their high immunogenicity, are thought to play a promising role in the development of immunodiagnostic reagents and vaccines. To enhance the antigenic and immunogenic properties of these proteins, fragments of the mpt83 and mpt63 genes were fused in tandem. In this article we present an effective method for the MPT63-MPT83 fusion product purification by metal-affinity chromatography and in vitro refolding. Our results demonstrate that the antigenic properties of the recombinant proteins obtained are comparable to their native analogues. The anti-rMPT63 and anti-rMPT83 sera were found to be highly reactive against the rMPT63-MPT83 fusion protein, which suggests that the fusion protein retains the antigenic properties of the parent proteins. Our results may potentially contribute to the development of improved diagnostic tools or vaccines against human and/or cattle tuberculosis.
- MeSH
- antigeny bakteriální * analýza genetika imunologie MeSH
- bakteriální proteiny * analýza genetika imunologie MeSH
- buněčná inkluze MeSH
- chromatografie MeSH
- epitopy imunologie MeSH
- Mycobacterium tuberculosis * genetika imunologie MeSH
- rekombinantní fúzní proteiny analýza biosyntéza MeSH
- rekombinantní proteiny biosyntéza terapeutické užití MeSH
- renaturace proteinů MeSH
- sbalování proteinů MeSH
- syntetické vakcíny genetika imunologie MeSH
- techniky amplifikace nukleových kyselin MeSH
- techniky in vitro MeSH
- western blotting MeSH
Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4U/mL of wet matrix.
- MeSH
- bioreaktory MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- enteropeptidasa biosyntéza chemie genetika MeSH
- enzymy imobilizované chemie genetika metabolismus MeSH
- histidin genetika metabolismus MeSH
- klonování DNA MeSH
- lidé MeSH
- Pichia genetika metabolismus MeSH
- rekombinantní fúzní proteiny biosyntéza chemie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 microM; for Staphylococcus aureus 2.31 microM, and for Enterococcus faecalis 5.54 microM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of beta-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.
- MeSH
- bakteriofág T7 genetika MeSH
- Candida albicans účinky léků MeSH
- DNA řízené RNA-polymerasy biosyntéza genetika MeSH
- Enterococcus faecalis účinky léků MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- kationické antimikrobiální peptidy biosyntéza genetika izolace a purifikace MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- promotorové oblasti (genetika) MeSH
- rekombinantní fúzní proteiny biosyntéza genetika izolace a purifikace MeSH
- Staphylococcus aureus účinky léků MeSH
- virové proteiny biosyntéza genetika MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
FerB is a flavoenzyme capable of reducing quinones, ferric complexes and chromate. Its expression in Escherichia coli as a hexahistidine fusion resulted in a functional product only when the tag was placed on the C-terminus. The molecular mass values estimated by gel permeation chromatography were compatible with the existence of either dimer or trimer, whereas the light scattering data, together with cross-linking experiments that yielded exclusively monomer and dimer bands on dodecyl sulfate-polyacrylamide gels, strongly supported a dimeric nature of both native and tagged form of FerB. These two proteins also exhibited almost identical secondary structure as judged by Fourier transform infra red spectrometry. The presence of tag, however, shifted the temperature of thermal inactivation as well as the thermal denaturation curve towards lower temperatures. Despite somewhat lower thermal stability, the fusion protein is considered a better candidate for crystallization than the wild-type one due to a more negative value of its second optical viral coefficient.
- MeSH
- diferenciální skenovací kalorimetrie MeSH
- Escherichia coli genetika MeSH
- Fourierova analýza MeSH
- histidin genetika chemie metabolismus MeSH
- multimerizace proteinu MeSH
- NADH, NADPH oxidoreduktasy biosyntéza genetika chemie metabolismus MeSH
- NADP metabolismus MeSH
- oligopeptidy genetika chemie metabolismus MeSH
- Paracoccus denitrificans enzymologie genetika MeSH
- rekombinantní fúzní proteiny biosyntéza genetika chemie metabolismus MeSH
- sekundární struktura proteinů MeSH
- stabilita enzymů MeSH
- teplota MeSH
- Publikační typ
- práce podpořená grantem MeSH
