Larisa A. Chupova

Crystal growth of phosphopantetheine adenylyltransferase, carboxypeptidase t, and thymidine phosphorylase on the international space station by the capillary counter-diffusion method

Crystals of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis, thymidine phosphorylase from Escherichia coli, carboxypeptidase T from Thermoactinomyces vulgaris and its mutant forms, and crystals of complexes of these proteins with functional ligands and inhibitors were grown by the capillary counter-diffusion method in the Japanese Experimental Module Kibo on the International Space Station. The high-resolution X-ray diffraction data sets suitable for the determination of high-resolution three-dimensional structures of these proteins were collected from the grown crystals on the SPring-8 synchrotron radiation facility. The conditions of crystal growth for the proteins and the data-collection statistics are reported. The crystals grown in microgravity diffracted to a higher resolution than crystals of the same proteins grown on Earth.

Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system

Chemical-enzymatic synthesis of human Epidermal Growth Factor (hEGF) cDNA has been performed, following by cloning into expression vector pTWIN1 (New England Biolabs). The resulting recombinant fusion protein expressed in Escherichia coli consisted of the N-terminal chitin-binding domain, mini-intein Ssp dnaB domain and hEGF polypeptide at the C-terminus. In this construct, mini-intein Ssp dnaB played a role of catalytically active subunit capable under certain conditions of autocatalytic cleavage resulting in separation of the target protein. As the hybrid protein had several cysteins in its sequence-one in chitin-binding domain, one in mini-intein and six in hEGF, it was necessary to work out optimal scheme for refolding and purification of the recombinant hEGF. As a result of this work, two schemes of the recombinant hEGF purification have been developed: according to the first scheme, the recombinant protein with reduced cysteins is bound to the chitin column, the hEGF is cleaved off and eluted, and then refolded to form appropriate cystein bridges. In the second scheme, the entire hybrid protein is first refolded to form disulfide bonds and then loaded to affinity resin; the recombinant hEGF is cleaved off and eluted in its native state. In spite of the fact that the first scheme is more common and suitable for a variety of recombinant proteins, in case of recombinant hEGF, the second scheme proved to be more productive and cost-effective.

X-ray study of the conformational changes in the molecule of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis during the catalyzed reaction.

Structures of recombinant phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium tuberculosis (PPATMt) in the apo form and in complex with the substrate ATP were determined at 1.62 and 1.70 Å resolution, respectively, using crystals grown in microgravity by the counter-diffusion method. The ATP molecule of the PPATMt-ATP complex was located with full occupancy in the active-site cavity. Comparison of the solved structures with previously determined structures of PPATMt complexed with the reaction product dephosphocoenzyme A (dPCoA) and the feedback inhibitor coenzyme A (CoA) was performed using superposition on C(α) atoms. The peculiarities of the arrangement of the ligands in the active-site cavity of PPATMt are described. The conformational states of the PPAT molecule in the consequent steps of the catalyzed reaction in the apo enzyme and the enzyme-substrate and enzyme-product complexes are characterized. It is shown that the binding of ATP and dPCoA induces the rearrangement of a short part of the polypeptide chain restricting the active-site cavity in the subunits of the hexameric enzyme molecule. The changes in the quaternary structure caused by this rearrangement are accompanied by a variation of the size of the inner water-filled channel which crosses the PPAT molecule along the threefold axis of the hexamer. The molecular mechanism of the observed changes is described.

Production and Purification of Recombinant Human Glucagon Overexpressed as Intein Fusion Protein in Escherichia coli

Chemico-enzymatic synthesis and cloning in Esherichia coli of an artificial gene coding human glucagon was performed. Recombinant plasmid containing hybrid glucagons gene and intein Ssp dnaB from Synechocestis sp. was designed. Expression of the obtained hybrid gene in E. coli, properties of the formed hybrid protein, and conditions of its autocatalytic cleavage leading to glucagon formation were studied.

Preparation of the Crystal Complex of Phosphopantetheine Adenylyltransferase from Mycobacterium tuberculosis with Coenzyme A and Investigation of Its Three-Dimensional Structure at 2.1-Å Resolution

Recombinant phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis (PPAT Mt), which was produced by a high-producing strain and purified to 99%, was used for the crystal growth of the complex of the enzyme with coenzyme A (CoA). Crystals suitable for X-ray diffraction study were obtained by cocrystallization. The crystals belong to sp. gr. R32 and have the unit-cell parameters a = b = 98.840 Å, c = 112.880 Å, α = β = 90.00°, and γ = 120.00°. The three-dimensional structure of the complex was determined based on X-ray diffraction data collected from the crystals to 2.1 Å resolution and refined to Rf = 22.7% and Rfree = 25.93%. Active-site bound coenzyme A was found, and its nearest environment was described. The conformational changes of the enzyme due to ligand binding were revealed. The binding of CoA by tuberculosis phosphopantetheine adenylyltransferase was characterized by comparing the structures of the title complex to a similar complex of PPAT from E. coli (PPAT Ec).

Three-Dimensional Structure of Phosphopantetheine Adenylyltransferase from Mycobacterium Tuberculosis in the Apo Form and in Complexes with Coenzyme A and Dephosphocoenzyme A

Crystals of phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium tuberculosis in the apo form and in complexes with coenzyme A (PPAT/CoA) and dephosphocoenzyme A (PPAT/dPCoA) were grown in microgravity by the capillary counter-diffusion method. The structures of PPAT Mt in the apo form and in complexes with ligands were solved based on the X-ray diffraction data collected from the grown crystals. The crystal structures were refined at 1.76, 1.59, and 1.59 Å resolution to Rf factors of 0.175, 0.159, and 0.157 and Rfree of 0.224, 0.208, and 0.206 for PPAT, PPAT/CoA, and PPAT/dPCoA, respectively. The atomic coordinates of the structures were deposited in the Protein Data Bank (PDB ID: 3RFF, 3RHS, and 3RBA). In these structures, the ligand-binding sites were determined, the environment of these sites was characterized, and the conformational changes accompanying the ligand binding were analyzed.

Recombinant Thymosin α1

An artificial gene encoding thymosin α1 was obtained by chemoenzymatic synthesis and cloned into Escherichia coli. An expressing recombinant plasmid containing the hybrid protein gene, which encodes amino acid sequences of thymosin α1 and the Saccharomycescerevisiae intein Sce VMA, was constructed. The expression of the hybrid protein from the resulting hybrid gene in E. coli, the properties of the resulting hybrid protein, and the conditions for its nonenzymatic cleavage to thymosin α1 were studied.

Production of Recombinant Oxytocin Through Sulfitolysis of Inteincontaining Fusion Protein

An artificial gene consisting of seven copies of an oxytocinoyl-lysine encoding sequence arranged in a tandem was synthesized and inserted downstream of the SspDnaB intein gene in a pTWIN1 plasmid. The corresponding fusion protein Dnab-7oxy contained 16 cysteine residues and formed inclusion bodies when expressed in E. coli. The standard protocol involving solubilization of the fusion protein and its autocatalytic cleavage on a chitin resin was not effective because of a very low yield of the cleavage reaction. Attempts to perform a refolding of the intein part of the fusion protein in solution were also unsuccessful because of a high level of protein aggregation. Sulfitolysis of cysteine residues is known to increase a solubility of proteins and peptides. Therefore we suggested a one-step approach that combines solubilization of inclusion bodies and sulfitolysis of a hybrid protein. The fusion protein was completely reduced and solubilized in 8M urea at pH 9.0 in the presence of sodium sulfite and sodium tetrathionate. The sulfitized protein was loaded onto a chitin column, an efficient cleavage was induced by a pH shift from 9.0 to 6.5, and seven successively connected oxytocinoyl- lysine units were released. The heptamer was subjected to trypsinolysis yielding sulfitized monomers of oxytocinoyllysine. Oxytocinoyl-lysine was refolded as described previously and treated by carboxypeptidase B to form the oxytocinic acid. The target oxytocin amide was then synthesized via methyl ester intermediate. Using this approach 6 mg of recombinant oxytocin can be obtained from 1 g of biomass.

Crystallization and preliminary X-ray diffraction study of phosphopantetheine adenylyltransferase from M. tuberculosis crystallizing in space group P32

Crystals of M. tuberculosis phosphopantetheine adenylyltransferase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 2.00-Å resolution. The crystals belong to sp. gr. P32 and have the following unit-cell parameters: a = b = 106.47 Å, c = 71.32 Å, α = γ = 90°, β = 120°. The structure was solved by the molecular-replacement method. There are six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is a biologically active form of phosphopantetheine adenylyltransferase from M. tuberculosis.

Recombinant fragment 44–77 of the pigment epithelium-derived factor prevents the development of the pathological cornea neovascularization

The pigment epithelium-derived factor (PEDF), a secreted 50 kDa glycoprotein, is one of the most potent endogenous inhibitors of angiogenesis. The fragment 44–77 of PEDF possesses the antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of diseases of visual organs accompanied by pathological neovascularization. An effective biotechnological method for the large-scale production of the PEDF (44–77) fragment as part of the fusion protein with the SspDnaB intein has been developed. The hybrid protein was produced in Escherichia coli bacterial cells in the form of inclusion bodies, solubilized, and subjected to autocatalytic cleavage with the release of the PEDF (44–77) fragment (reaction yield 77%). The target peptide was separated from the intein by tangential ultrafiltration. The final purification of PEDF (44–77) was performed by reversed-phase HPLC. The yield of the target peptide (purity 99%) was 65 mg per 1 l of culture. The antiangiogenic activity of the peptide was confirmed in vitro using mouse endothelial cells SVEC-4-10. It was found that the preparation at a concentration of 1 nM suppresses the proliferation of endothelial cells by 53% and inhibits the formation of tube-like structures by endothelial cells. The ability of the recombinant PEDF (44–77) to block the initial stages of angiogenesis was shown using an experimental model of rabbit corneal neovascularization.