Heterologous Expression of Codon-Optimized Azurin Transferred by Magnetofection Method in MCF-10A Cells.

Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH2) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects.

Affinity-based and in a single step purification of recombinant horseradish peroxidase A2A isoenzyme produced by Pichia pastoris.

Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests, probe-based test techniques, removal of phenolic pollutants from wastewater and organic synthesis. HRP is found in the root of the horseradish plant as a mixture of different isoenzymes, and it is very difficult to separate these enzymes from each other. In this regard, recombinant production is a very advantageous method in terms of producing the desired isoenzyme. This study was performed to produce HRP A2A isoenzyme extracellularly in Pichia pastoris and to purify this enzyme in a single step using a 3-amino-4-chloro benzohydrazide affinity column. First, codon-optimized HRP A2A gene was amplified and inserted into pPICZαC. So, obtained pPICZαC-HRPA2A was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized recombinant DNA and a yeast clone was cultivated for extracellular recombinant HRP A2A (rHRP A2A) enzyme production. Then, the purification of this enzyme was performed in a single step by affinity chromatography. The molecular mass of purified rHRP A2A enzyme was found to be about 40 kDa. According to characterization studies of the purified enzyme, the optimum pH and ionic strength for the rHRP A2A isoenzyme were determined to be 6.0 and 0.04 M, respectively, and o-dianisidine had the highest specificity with the lowest Km and Vmax values. Thus, this is an economical procedure to purify HRP A2A isoenzyme without time-consuming and laborious isolation from an isoenzyme mixture.

Comparative heating efficiency and cytotoxicity of magnetic silica nanoparticles for magnetic hyperthermia treatment on human breast cancer cells.

Magnetic hyperthermia (MHT) is a promising treatment for a variety of cancers due to its ability to increase the sensitivity of cells to other treatments, such as chemotherapy. Superparamagnetic nanoparticles (MNPs) were used for MHT treatment due to their heat generation ability under an AC magnetic field (AMF). In this study, iron oxide and zinc-doped iron oxide MNPs were produced and modified with silica to obtain eleven different types (MSNP-I to -XI) of magnetic silica nanoparticles (MSNPs). The MSNPs which show the highest heating capacity were selected to investigate their MHT ability on non-tumourigenic MCF-10A and tumourigenic MCF-7 cell lines. The cytotoxicity results indicated that the size, the content of the magnetic core and silica coating thickness were important in the heating capacity of MSNPs under AMF. After MHT treatment, selected MSNPs showed limited cytotoxicity on MCF-10A, but significant cell death on MCF-7. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03377-y.

Constitutive and extracellular expression of pectin methylesterase from Pectobacterium chrysanthemi in Pichia pastoris.

Pectin methylesterase (PME) which is widely used in the cosmetic, food and pharmaceutical industries catalyses the hydrolysis of the methyl ester of pectin to yield methanol and free carboxyl groups. This study was performed to produce active pectin methylesterase (PME) extracellularly from Pectobacterium chrysanthemi in Pichia pastoris. Firstly, pGKBα was constructed for the secretion of heterologous protein. After it was cloned in Escherichia coli cells and the sequence was affirmed, PME gene was inserted into pGKBα. So, pGKBα-PME carried the PME gene in correct position was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized pGKBα-PME and six transformants were cultivated for recombinant PME production. It was observed that one of them had a high-capacity secretion of active PME. The molecular mass of extracellular PME enzyme was found to be about 59 kDa. The PME enzyme from P. chrysanthemi was produced by P. pastoris for the first time in this study. This recombinant enzyme might be produced in a large scale and also purified from the culture medium. Then, the purified enzyme might be used for clarification and increasing yield of juice in food industrial applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03291-3.

Extracellular production of azurin from Pseudomonas aeruginosa in the presence of Triton X-100 or Tween 80.

Azurin which is a bacterial secondary metabolite has attracted much attention as potential anticancer agent in recent years. This copper-containing periplasmic redox protein supresses the tumor growth selectively. High-level secretion of proteins into the culture medium offers a significant advantage over periplasmic or cytoplasmic expression. The aim of this study was to investigate the effect of nonionic surfactants on the expression of the Pseudomonas aeruginosa azurin. Different concentrations of Triton X-100 and Tween 80 were used as supplements in growth media and extracellular azurin production was stimulated by both surfactants. According to western blot analysis results, in the presence of Triton X-100, maximum azurin expression level was achieved with 96 h of incubation at 1% concentration, and 48 h at 2% concentration. On the other hand, maximum azurin expression level was achieved in the presence of 1% Tween 80 at 72 h incubation. This study suggested for the first time a high level of azurin secretion from P. aeruginosa in the presence of Triton X-100 or Tween 80, which would be advantageous for the purification procedure.

Magnetic nanoparticle mediated-gene delivery for simpler and more effective transformation of Pichia pastoris.

The introduction of exogenous DNA into a cell can be used to produce large quantities of protein. Here, we describe a novel gene delivery method for Pichia pastoris based on recombinant DNA delivery using magnetic nanoparticles (MNPs) under magnetic forces. For this purpose, a linear plasmid (pGKB-GFP) containing the Green Fluorescent Protein (GFP) gene is loaded on polyethyleneimine-coated iron oxide (Fe3O4@PEI) MNPs at doses that are non-toxic to the yeast cells. The pGKB-GFP loaded MNPs combined with enhancer PEI (Fe3O4@PEI + pGKB-GFP + PEI) are directly transferred to non-competent cells. An effective GFP expression was observed by the selection of antibiotic-resistant yeast cells and heterologous gene integration into the P. pastoris genome was provided. This method, which is very simple, effective, and advanced equipment-free compared to traditional methods, uses smaller amounts of DNA and the process can be performed in a shorter time. The suggested method might also be adapted for the transformation of other yeast species.

Heterologous expression of azurin from Pseudomonas aeruginosa in the yeast Pichia pastoris.

Azurin, which is a bacterial secondary metabolite has been attracted as a potential anticancer agent in recent years because induced death of cancer cells and inhibited their growth. In this study, the production of azurin under the control of the alcohol oxidase promoter which is frequently used in the Pichia pastoris expression system was performed. The azurin gene amplified from Pseudomonas aeruginosa genomic DNA and inserted into the pPICZαA was cloned in Escherichia coli cells. Then, a linearized recombinant vector was transferred to the P. pastoris X-33 cells. Antibiotic resistance test and colony PCR were performed for the selection of multicopy transformants. Protein expression capacities of selected transformants were compared at the end of 48 h incubation. Both extracellular and intracellular protein expressions were observed in all of them by Western blot analysis. The relative expression levels of both intracellular and extracellular protein that belongs to the first clone were higher than the others. On the other hand, it was seen that the 4th clone had the highest protein secretion ability. The molecular mass of the extracellular azurin protein which is produced by recombinant clones was found to be about 20 kDa. This is the first report on azurin expression in P. pastoris.

Bone turnover in chronic otitis media with bone destruction.

PURPOSE: This study aims to determine the relationship between bone destruction and bone turnover markers in patients with chronic otitis media (COM). METHODS: Subjects with COM were divided into two groups: those with and without bone destruction. Thirty-seven patients were included in the group with bone destruction; 30 patients were included in the group without bone destruction. The enzyme values were evaluated. RESULTS: There was no difference between the two groups in terms of enzyme levels of serum and urine. However, the osteocalcin, which is a bone formation marker, and the C-terminal telopeptide of type I collagen marker, which is bone destruction marker, were found to be lower in the group with bone destruction than the group without bone destruction. CONCLUSION: Data obtained in the present study suggest that the pressure necrosis theory and acid lysis theory provide the most valid explanations of bone destruction. However, the data provide limited preliminary information to clarify this mechanism.