Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor.

Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods.

Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P.

Immobilization of His-tagged kinase JAK2 onto the surface of a plasmon resonance gold disc modified with different copper (II) complexes.

New surface plasmon resonance (SPR) sensing platforms which consists of copper (II) complexes of a pentetic acid thiol ligand (DPTA-Cu(II)) and of a thiol derivative of dipyrromethene (DPM-Cu(II) created on the surface of gold SPR disc were applied to oriented immobilization of His-tagged Janus kinase 2 (GST-His6-JAK2). This method is based on the covalent bond formation between histidine from a His-tag chain of a protein and Cu(II) centres from the complexes. The kinetic and thermodynamic parameters of the oriented immobilization of GST-His6-JAK2 protein to DPTA-Cu(II) and DPM-Cu(II) complexes attached to the Au surface of a SPR disc were discussed.

Analogs of cinnamic acid benzyl amide as nonclassical inhibitors of activated JAK2 kinase.

Scaffold-based analogs of cinnamic acid benzyl amide (CABA) exhibit pleiotropic effects in cancer cells, and their exact molecular mechanism of action is under investigation. The present study is part of our systemic analysis of interactions of CABA analogs with their molecular targets. These compounds were shown to inhibit Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and JAK2/signal transducer and activator of transcription 5 (STAT5) signaling and thus are attractive scaffolds for anticancer drug design.

Development of novel molecular probes of the Rio1 atypical protein kinase.

The RIO kinases are essential protein factors required for the synthesis of new ribosomes in eukaryotes. Conserved in archaeal organisms as well, RIO kinases are among the most ancient of protein kinases. Their exact molecular mechanisms are under investigation and progress of this research would be significantly improved with the availability of suitable molecular probes that selectively block RIO kinases.

N-linked glycosylation of G. mellonella juvenile hormone binding protein - comparison of recombinant mutants expressed in P. pastoris cells with native protein.

Juvenile hormone (JH) regulates insect growth and development. JH present in the hemolymph is bound to juvenile hormone binding protein (hJHBP) which protects JH from degradation. In G.

Immunosensor incorporating anti-His (C-term) IgG F(ab') fragments attached to gold nanorods for detection of His-tagged proteins in culture medium.

Immunosensors based on gold electrodes (electrochemical) or gold discs (optical) modified with 1,6-hexanedithiol, gold nanorods and Anti-His (C-term) monoclonal antibody F(ab') fragment are described. The antigen detected by the sensing platform is a recombinant histidine-tagged silk proteinase inhibitor (rSPI2-His(6)). Electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) techniques were used as methods for detection of the antigen.

Comparison of electrochemical immunosensors based on gold nano materials and immunoblot techniques for detection of histidine-tagged proteins in culture medium.

In this work, the direct electrochemical determination of poly-histidine tagged proteins using immunosensor based on anti-His (C-term) antibody immobilized on gold electrodes modified with 1,6-hexanedithiol, gold colloid particles or gold nanorods is described. The recombinant histidine-tagged silk proteinase inhibitor protein (rSPI2-His(6)) expressed in Pichia system selected as antigen for this immonosensor. An electrochemical impedance spectroscopy was used as label free detection technique for immune conjugation.

Electrochemical impedance spectroscopy for the study of juvenile hormones-recombinant protein interactions.

The interactions of recombinant juvenile hormone binding protein (His8-rJHBP) with juvenile hormones (JHs), methoprene and farnesol have been studied with electrochemical impedance spectroscopy (EIS). The protein was immobilized on the dodecanethiol (DDT) modified gold electrodes. Each step of electrode modification has been confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).

Structurally unique recombinant Kazal-type proteinase inhibitor retains activity when terminally extended and glycosylated.

Recombinant derivatives of the Kazal-type serine proteinase inhibitor GmSPI2 (36 amino acid residues), which is a component of insect silk, were prepared in the expression vector Pichia pastoris. The rhSPI2 had a C-terminal hexahistidine tag attached to the GmSPI2 sequence, rtSPI2 was extended with GluAlaAla at the N-terminus, and rfSPI2 included this N-terminal extension and a C-terminal tail of 22 residues (myc epitope and hexahistidine). A portion of the secreted rfSI2 was O-glycosylated with a trimannosyl or hexamannosyl.

Positions of disulfide bonds and N-glycosylation site in juvenile hormone binding protein.

The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues. It contains four Cys residues forming two disulfide bridges. In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G.

Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris.

Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis.

Hormonal regulation of the expression of two storage proteins in the larval fat body of the greater wax moth (Galleria mellonella).

During larval development of the greater wax moth, Galleria mellonella, genes of storage proteins LHP76 and LHP82 are tissue- and stage-specifically expressed. In this study, hormonal regulation of this expression has been investigated in vivo. Messenger RNAs of the juvenile hormone (JH-suppressible) Lhp82 gene are present only during the feeding period of the final larval instar, suggesting that a high level of JH during earlier stages prevents its expression and that a small rise in JH titer observed on day 8 of the final larval instar is responsible for the rapid shut-off of its transcription.