Barbara Kludkiewicz

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. The gold nanorods modified electrode layer showed better analytical response than gold nano particles. The linear calibration range was observed between 10 pg/ml and 1 ng/ml with limit of detection 5 pg/ml (S/N=3). Up to four successive assay cycles with retentive sensitivity were achieved for the immunosensors regenerated with 0.2M glycine-HCl buffer, pH 2.8. The performance of this immnosensor were compared with immuoblotting techniques.

Expression of larval hemolymph proteins (Lhp) genes and protein synthesis in the fat body of greater wax moth (Galleria mellonella) larvae during diapause

When one-day-old, last instar Galleria mellonella larvae are exposed to 18 degrees C they enter diapause and cease further development for several months. During diapause a group of proteins (72-84 kDa) synthesized in the fat body and secreted into the hemolymph is markedly elevated. Partial sequencing of the N-terminus of two proteins from this group confirmed their identity with larval hemolymph proteins (LHP) belonging to the family of hexameric storage proteins. The expression of two Lhp genes of known sequence (Lhp76 and Lhp82) were monitored in both diapausing and non-diapausing individuals. The expression of both genes and subsequent synthesis of the proteins (LHP76 and LHP82) is maintained until at least 90-100 days of diapause.

Glutathione–ascorbic acid redox cycle and thioredoxin reductase activity in the digestive tract of Leptinotarsa decemlineata (Say)

In view of the antioxidant role of glutathione (GSH) and ascorbic acid (AA), we have examined capacity of the GSH-AA redox cycle in relation to oxidative stress effects in the midgut of the Colorado potato beetle Leptinotarsa decemlineata. Adult gut harbors a higher capacity to cope with oxidative stress than the larval gut. Protein carbonylation was pronounced in the wall of anterior larval midgut and was generally lower in the food digest than in the gut wall. Restriction of oxidative stress effects in anterior gut lumen manifested by lipid peroxidation and protein carbonylation is interpreted as a mechanism favoring digestion and absorption in the posterior midgut. Presence of high GSH in the posterior midgut and AA in both posterior and anterior midguts of adults points to higher utility of the GSH-AA redox system in limiting oxidative stress to manageable levels. The presence, gene expression and activity of thioredoxin reductase (TrxR) were demonstrated for the first time in L. decemlineata which was markedly higher in the anterior than in the posterior midgut in both stages. It is probably central to the maintenance of reduced GSH levels in the whole gut, despite a GSSG/2GSH redox potential tending towards oxidizing ranging from -183.5 to -124.4mV. Glutathione-dehydroascorbate reductase (G(DHA)R) activity was markedly augmented in adult gut compared with larva, pointing to a more efficient conversion of dehydroascorbate (DHA) to AA. Also, ascorbate peroxidase (APOX) activity was significantly elevated in all gut compartments of adult except the wall of posterior midgut. The results emphasize the potential importance and role of the GSH-AA redox cycle as a defense strategy against oxidative stress in the gut of L. decemlineata.

Low molecular weight silk proteins in Galleria mellonella

Abstract Galleria cocoon proteins have been extracted by different solubilizing agents. Nine protein bands were observed by gel electrophoresis, with molecular weights ranging from 18 to 420 kD. Three silk proteins of 24, 29 and 30 kD were extracted only in the presence of β-mercaptoethanol, suggesting that they are covalently linked by disulfide bonds to the large fibroin. They are likely to be the products of the highly abundant mRNA of the posterior silk gland cells. In vitro translation analysis of this mRNA yielded 24, 29 and 30 kD proteins. Thus, as in Bombyx , the Galleria silk is composed of several subunits, including fibroin and low molecular weight polypeptides. However, the genes coding for fibroin or low molecular weight silk proteins in Bombyx and Galleria do not show nucleotide base homology.

The effect of 20-hydroxyecdysone on expression of genes coding for low molecular weight silk proteins of Galleria mellonella

Abstract The low molecular weight Galleria silk proteins of 24 and 29–30 kDa are coded by the 1100 nucleotides long mRNAs of the posterior silk gland (PSG) cells. The synthesis of these proteins starts during the first 2 days of the last instar when the endogenous ecdysteroid titre is very low. The synthesis of 24 kDa protein precedes the synthesis of 30 kDa protein. PSGs from day 1 last instar larvae are sensitive to exogenous 20-hydroxyecdysone (20-HE) in vitro at 0.5 μg/ml concentration causing an increase of 1100 nucleotides long mRNAs. The electrophoretic analysis of the in vitro labelling proteins indicates that 20-HE may induced the synthesis of 30 kDa protein. Juvenile hormone II in vitro suppresses the stimulatory effect of 20-HE. PSGs from day 3 last instar larvae are insensitive to exogenous 20-HE in vitro .

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. Application of 1micro g of JH analog (fenoxycarb) specifically inhibits expression of Lhp82, whereas Lhp76 mRNAs remain at the same level. 20-hydroxyecdysone (20HE) does not exert any inhibitory effects on transcription of Lhp genes when injected in a dose of 0.5 or 1.5 micro g per individual, regardless of larval age. However, the same dose of 20HE significantly lowers the rate of LHPs synthesis within the fat body and completely blocks secretion of LHPs into the hemolymph. Therefore, we propose that 20HE inhibits the synthesis of storage proteins and their secretion without altering the level of mRNAs.

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. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.

Functional conservation and structural diversification of silk sericins in two moth species.

Sericins are hydrophilic structural proteins produced by caterpillars in the middle section of silk glands and layered over fibroin proteins secreted in the posterior section. In the process of spinning, fibroins form strong solid filaments, while sericins seal the pair of filaments into a single fiber and glue the fiber into a cocoon. Galleria mellonella and the previously examined Bombyx mori harbor three sericin genes that encode proteins containing long repetitive regions. Galleria sericin genes are similar to each other and the protein repeats are built from short and extremely serine-rich motifs, while Bombyx sericin genes are diversified and encode proteins with long and complex repeats. Developmental changes in sericin properties are controlled at the level of gene expression and splicing. In Galleria , MG-1 sericin is produced throughout larval life until the wandering stage, while the production of MG-2 and MG-3 reaches a peak during cocoon spinning.

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. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and R free of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β2αβ fold characteristic for Kazal-family serine proteinase inhibitors.

The effect of transcription inhibitors on early development of the avian embryo

The effect of transcription [actinomycin D, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), alpha-amanitin] and translation inhibitors (cycloheximide, puromycin) on quail embryo development was investigated under in vitro conditions. The gastrulation process seemed to proceed normally in the presence of transcription inhibitors in the medium but the translation inhibitors stopped development and caused complete degeneration of the embryos.