Increasing productivity of arylsulfatase B-producing cell line by coexpression of formylglycine-generating enzyme

Mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome) is an orphan genetic disease caused by deficiency of the lysosomal enzyme arylsulfatase B (ASB). The need to develop a highly productive cell line for the production of recombinant ASB, is behind the concept and relevance of this study. The most promising approach seems to be the development of CHO producer cell lines coexpressing the target ASB enzyme and an auxiliary formylglycine-generating enzyme (FGE). At the same time, it is important from a practical perspective to have the possibility of cultivating producer cell lines as suspensions free of serum or other components of animal origin. The aim of the study was to develop highly productive cell lines for the production of recombinant ASB by coexpression of the auxiliary FGE. Materials and methods: a suspension CHO cell line was used in the study. CHO cells were transfected by electroporation using the MaxCyte STX system. Monoclonal cell lines were obtained with the help of the Cell Metric system. Enzyme-linked immunosorbent assay was used for determination of ASB concentration in the culture fluid. Culture fluid samples were analysed using polyacrylamide gel electrophoresis and Western blotting. The mRNA level was measured by real-time polymerase chain reaction. Results: producer cell lines coexpressing the target ASB enzyme and auxiliary FGE were obtained. An increase in the yield of the active target ASB enzyme from 2 to 100 mg/L was achieved by selecting the optimal ratio of plasmids during transfection. The highest yield of the target ASB enzyme was achieved at the 90:10 ratio (%) of plasmids encoding the ASB and FGE genes, respectively. Conclusions: the authors developed highly productive cell lines for the production of recombinant ASB, which coexpress the target and auxiliary enzymes. The coexpression of ASB and FGE improves the growth and production characteristics of the cell line, probably due to the modification of the ASB active site. The obtained results will help resolve the problem of low enzyme yield, which is typical of this class of medicines

Увеличение продуктивности клеточной линии-продуцента арилсульфатазы B за счет коэкспрессии формилглицин-генерирующего фермента

Mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome) is an orphan genetic disease caused by deficiency of the lysosomal enzyme arylsulfatase B (ASB). The need to develop a highly productive cell line for the production of recombinant ASB, is behind the concept and relevance of this study. The most promising approach seems to be the development of CHO producer cell lines coexpressing the target ASB enzyme and an auxiliary formylglycine-generating enzyme (FGE). At the same time, it is important from a practical perspective to have the possibility of cultivating producer cell lines as suspensions free of serum or other components of animal origin. The aim of the study was to develop highly productive cell lines for the production of recombinant ASB by coexpression of the auxiliary FGE. Materials and methods: a suspension CHO cell line was used in the study. CHO cells were transfected by electroporation using the MaxCyte STX system. Monoclonal cell lines were obtained with the help of the Cell Metric system. Enzyme-linked immunosorbent assay was used for determination of ASB concentration in the culture fluid. Culture fluid samples were analysed using polyacrylamide gel electrophoresis and Western blotting. The mRNA level was measured by real-time polymerase chain reaction. Results: producer cell lines coexpressing the target ASB enzyme and auxiliary FGE were obtained. An increase in the yield of the active target ASB enzyme from 2 to 100 mg/L was achieved by selecting the optimal ratio of plasmids during transfection. The highest yield of the target ASB enzyme was achieved at the 90:10 ratio (%) of plasmids encoding the ASB and FGE genes, respectively. Conclusions: the authors developed highly productive cell lines for the production of recombinant ASB, which coexpress the target and auxiliary enzymes. The coexpression of ASB and FGE improves the growth and production characteristics of the cell line, probably due to the modification of the ASB active site. The obtained results will help resolve the problem of low enzyme yield, which is typical of this class of medicines.Мукополисахаридоз VI типа (синдром Марото–Лами) — орфанное генетическое заболевание, которое связано с дефицитом лизосомального фермента арилсульфатазы В. Актуальность исследования связана с необходимостью разработки высокопродуктивной клеточной линии-продуцента рекомбинантного фермента арилсульфатазы В. Наиболее перспективным подходом представляется создание клеточных линий-продуцентов, коэкспрессирующих целевой фермент арилсульфатазу В и вспомогательный формилглицин-генерирующий фермент на основе клеточной линии СНО. При этом большое практическое значение имеет возможность культивирования клеточных линий-продуцентов в виде суспензии, без использования сыворотки или других компонентов животного происхождения. Цель работы: разработка высокопродуктивных клеточных линий-продуцентов рекомбинантного фермента арилсульфатазы В за счет коэкспрессии вспомогательного формилглицин-генерирующего фермента. Материалы и методы: использовали суспензионную клеточную линию СНО. Трансфекцию клеток СНО проводили методом электропорации с использованием системы MaxCyte STX. Моноклональные клеточные линии получали с использованием системы Cell Metric. Концентрацию арилсульфатазы В в культуральной жидкости определяли методом иммуноферментного анализа. Образцы культуральной жидкости анализировали с применением электрофореза в полиакриламидном геле и вестерн-блота. Уровень мРНК измеряли методом полимеразной цепной реакции в режиме реального времени. Результаты: получены клеточные линии-продуценты, коэкспрессирующие целевой фермент арилсульфатазу В и вспомогательный формилглицин-генерирующий фермент. Достигнуто увеличение выхода активного целевого фермента арилсульфатазы В с 2 до 100 мг/л за счет подбора оптимального соотношения плазмид во время трансфекции. Наибольший выход целевого фермента арилсульфатазы В наблюдался при соотношении плазмид, кодирующих гены арилсульфатазы В и формилглицин-генерирующего фермента, равном 90:10 (%). Выводы: разработаны высокопродуктивные клеточные линии-продуценты рекомбинантного фермента арилсульфатазы В, коэкспрессирующие целевой и вспомогательный ферменты. Коэкспрессия арилсульфатазы В и формилглицин-генерирующего фермента приводит к улучшению ростовых и продукционных характеристик клеточной линии, что, по-видимому, обусловлено модификацией активного центра целевого фермента арилсульфатазы В. Полученные результаты позволят решить проблему низкого выхода фермента, характерную для препаратов подобного класса

Martensite-to-austenite reversion and recrystallization in cryogenically-rolled type 321 metastable austenitic steel

The annealing behavior of cryogenically-rolled type 321 metastable austenitic steel was established. Cryogenic deformation gave rise to martensitic transformation which developed preferentially within deformation bands. Subsequent annealing in the range of 600 C to 700 C resulted in reversion of the strain-induced martensite to austenite. At 800 C, the reversion was followed by static recrystallization. At relatively-low temperatures, the reversion was characterized by a very strong variant selection, which led to the restoration of the crystallographic orientation of the coarse parent austenite grains. An increase in the annealing temperature relaxed the variant-selection tendency and provided subsequent recrystallization thus leading to significant grain refinement. Nevertheless, a significant portion of the original coarse grains was found to be untransformed and therefore the fine-grain structure was fairly heterogeneous

EBSD characterization of cryogenically rolled type 321 austenitic stainless steel

Electron backscatter diffraction was applied to investigate microstructure evolution during cryogenic rolling of type 321 metastable austenitic stainless steel. As expected, rolling promoted deformation-induced martensitic transformation which developed preferentially in deformation bands. Because a large fraction of the imposed strain was accommodated by deformation banding, grain refinement in the parent austenite phase was minimal. The martensitic transformation was found to follow a general orientation relationship, {111}γ||{0001}ε||{110}α′ and 〈110〉γ||〈11-20〉ε||〈111〉α′, and was characterized by noticeable variant selection

“Treatment” pregnancy in captivity dangerous delusion

Availability of pharmaceuticals in this country has caused not only a self-patients, but the “treatment” of pregnancy. The effectiveness of some medications commonly prescribed in pregnancy has not been proved, and the number of drugs taken by pregnant sometimes exceeds all reasonable limits. The only possibility that avoids undesirable effects of medicines during pregnancy is to minimize their number by an informed, responsible and impartial approach to the appointment of each drug

Periodic variations in time of atmospheric alpha and beta radioactive nanoparticles

Here we present time variations data of radon emanations in the surface layer of the atmosphere, measured from January 2016 to June 2018. By means of spectral analysis method, we have analyzed time variations and showed the presence of existing natural variations, mechanisms of which were described by the authors in previous works. Authors measured the activity of natural beta-active radionuclides of radon’s daughter decay products (DDP) in the surface layer of the atmosphere. The measurements were performed with a beta-spectrometer “Progress” scintillation detector, which is located on the third floor of the Faculty of Physics and Technology at al-Farabi Kazakh National University (KazNU). Measurements of beta spectra were carried out with an exposure of at least for 2000 seconds and during the day up to 10 beta spectra were recorded. The built up time dependence of the integral values of the spectra shows that seasonal variations of natural beta-active radionuclides are manifested on the background of diurnal fluctuations. Thus, 1 day, 4 day, 7 day, 16 day and 30 day variations were found