RESUMO
The effect of recombinant spidroin (RS) hydrogel (HG) on anterior epithelial cells and keratocytes of the human cornea was studied in vitro. Corneal injuries are highly prevalent in developing countries according to the World Health Organization. Various technologies have recently been proposed to restore the damaged surface of the cornea. Use of biodegradable silk-based materials, including recombinant analogs of the spider silk protein spidroin, is an important avenue of research in the field of wound healing and corneal regeneration. Spidroins are well known for their optimal balance of strength and elasticity. Given their biological compatibility, lack of immunogenicity, and biodegradability, spidroins provide a biomaterial for tissue engineering and regenerative medicine. HGs based on RS rS2/12-RGDS were therefore tested for cytotoxicity toward isolated corneal epithelial cells and keratocytes with regard to possible changes in cell phenotype and migratory activity. A promising outlook and therapeutic potential were demonstrated for RS-based HGs.
Assuntos
Fibroínas , Humanos , Fibroínas/farmacologia , Fibroínas/genética , Seda/genética , Córnea , Materiais Biocompatíveis , Proliferação de CélulasRESUMO
Phospholipase A2 (PLA2) has found extensive use in industry. However, recombinant PLA2 production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA2 from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A2 protein were similar to those of the native Streptomyces violaceoruber PLA2 protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Fosfolipases A2/metabolismo , Proteínas de Bactérias/genética , Inteínas/genética , Inteínas/fisiologia , Fosfolipases/genética , Fosfolipases/metabolismo , Fosfolipases A2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Streptomyces/genética , Streptomyces/metabolismoRESUMO
The modified asparaginase Was79 was derived from the recombinant wild-type L-asparaginase of Wolinella succinogenes. The Was79 contains the amino acid substitutions V23Q and K24T responsible for the resistance to trypsinolysis and the N-terminal heparin-binding peptide KRKKKGKGLGKKR responsible for the binding to heparin and tumor K562 cells in vitro. When tested on a mouse model of Fischer lymphadenosis L5178Y, therapeutic efficacy of Was79 was significantly higher than that of reference enzymes at all single therapeutic doses used (125-8000 IU/kg). At Was79 single doses of 500-8000 IU/kg, the complete remission rate of 100 % was observed. The Was79 variant can be expressed intracellularly in E. coli as a less immunogenic formyl-methionine-free form at high per cell production levels.
Assuntos
Antineoplásicos/administração & dosagem , Asparaginase/genética , Asparaginase/metabolismo , Heparina/metabolismo , Leucemia L5178/tratamento farmacológico , Wolinella/enzimologia , Substituição de Aminoácidos , Animais , Antineoplásicos/farmacologia , Asparaginase/administração & dosagem , Asparaginase/farmacologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células K562 , Camundongos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Wolinella/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of recombinant plasmids has been constructed for expression of the hepatitis B viral surface antigen gene (HBsAg) under the control of the regulatory elements of the yeast acidic phosphatase gene (PHO5). The obtained plasmids possess the high mitotic and structural stability in the transformant yeast cells. The effect of different structural modifications of the vector on the level of HBsAg synthesis in yeasts has been studied. Optimal construction devoid of the bacterial DNA sequences and pre-S region has been selected.
Assuntos
Regulação Viral da Expressão Gênica , Genes Virais , Engenharia Genética , Antígenos de Superfície da Hepatite B/genética , Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Plasmídeos , Recombinação Genética , Transformação GenéticaRESUMO
The recombinant plasmids pPTX and pPGX were constructed, containing the sequences of yeast Ty retrotransposon and Drosophila element mdg4, correspondingly. Transformation of yeast by these plasmids lead to induction of reverse transcriptase activity associated with virus-like particles, containing only the sequences of Ty. The data obtained show that mdg4 is capable of expression in yeast and the products of its expression are used to form the yeast virus-like particles. The system described may be used to study the expression of different retrotransposons from various cells in yeast.
