A Novel Induction Strategy Based on Temperature and pH Optimization to Improve the Yield of Recombinant Streptokinase in Escherichia coli

Introduction:Streptokinase (SK) is used for thrombolytic therapy of acute myocardial infarction. Recombinant fermentation is the most common and cost-effective procedure in the production SK. Nutrients composition, as well as such fermentation variables as temperature and pH, can affect the production level of protein-based drugs expressed in E. coli. In the present study, optimized recombinant streptokinase (r-SK) production in E. coli in the form of inclusion body (IB), the effect of induction temperature and extracellular pH were evaluated.Methods and Results:To maximize the expression level of r-SK three different induction temperatures were used to obtain the optimal temperature for r-SK production. The effect of extracellular pH on the level of r-SK production was also studied and the optimal pH was determined. To this purpose, 8 fermentation processes were applied and followed by: Cell disruption, Isolation of IBs, determination of target protein (SDS-PAGE, Western-blot). The biological activity of r-SK obtained from optimal conditions was determined in comparison with reference standard using gel clot method.The highest amount of expression level and IBs formation were performed by combining two temperatures: 42˚C for the first two hours of induction followed by 39˚C at pH 6. The expression level of r-SK under these conditions shows an 11.6% increase in comparison with the control. Conclusions:Choosing appropriate cultivation conditions can increase IB formation and use its advantages to produce higher quantities of r-SK with appropriate biological activity

Design, synthesis, and characterization of thiol-decorated cross-linked graphene oxide framework for high-capacity Hg2+ ion adsorption

A 3D framework was effectively created by cross-linking graphene oxide nanosheets with 2,5-bis(((3-mercaptopropyl)dimethoxysilyl)oxy)terephthalohydrazide as a sulfur-containing linker, resulting in a successful synthesis of a novel chelating adsorbent. The resulting material showed remarkable efficiency in removing Hg2+ ions from polluted water, with a high adsorption capacity of 204.08 mg g−1 at pH 6 and 25 °C. The adsorption process followed a monolayer model described by the Langmuir isotherm, and the kinetics followed a pseudo-second-order model, indicating a chemical adsorption mechanism. The material demonstrated rapid adsorption and desorption, maintaining its performance even after multiple regeneration cycles. Overall, the synthesized 3D GOF stands out as an exceptional adsorbent due to its porous structure, efficient chelating functional groups, and robust efficacy in eliminating Hg2+ ions from contaminated water sources

Improvements in the Downstream Processing of rhGH production in Pichia pastoris: Optimization of hGH purification using activated carbon clarification in Pichia pastoris

Production of human proteins in Pichia pastoris has significant advantages. However, there is still need for improvement of various stages of its downstream processing like clarification and purification. In fact downstream processes are usually the most critical part of production of biotech products. This work aimed to evaluate the effect of two steps added to the downstream processes of human growth hormone (hGH) production in Pichia pastoris. Firstly, the effect of clarification, with activated carbon, on capture of hGH by ion exchange chromatography (IEC) was investigated. For this purpose, a clarification process using activated carbon was used to remove process contaminants like pigments. The clarified sample was applied to the IEC column and the recovery of hGH, following IEC, was assessed using SDS-PAGE, Bradford protein assay, and area under the curve (AUC). The obtained results showed that the AUC values were 2.81 and 5.61 for the with- and without-treatment samples, respectively. Protein recovery of clarified sample with activated carbon was 541 mg in comparison with 328 mg for the sample without treatment. The yield of IEC was also improved from 50.46% to 83.23% following treatment with activated carbon. Secondly, the effect of three concentrations of ammonium sulfate in the binding buffer on resolution of hGH upon elution on hydrophobic interaction chromatography (HIC) was investigated. Biological activity was used as the main criterion for evaluation of purified hGH using HIC. The obtained results indicated that by increasing the concentration of ammonium sulfate form 1 to 3 mol/L, resolution of hGH was improved, as the purified fraction using 3 mol/L of ammonium sulfate showed a specific activity of 3.1IU/mg. So, the results of the present study demonstrated that activated carbon is a promising candidate for efficient clarification of recombinant hGH and for improving the efficiency of the capture step. Therefore, it can be considered by biotech companies as a cost-effective and sustainable clarification procedure of recombinant proteins from high cell density cultures. This study also revealed that 3% ammonium sulfate has a positive effect on the separation of hGH variants with the desired biological activity

The fluctuation of APC gene in WNT signaling with adenine deletion of adenomatous polyposis coli, is associated in colorectal cancer

Colorectal cancer is one of the most important malignancies in the classification of gastrointestinal cancers. One of the predisposing factors at molecular level for this cancer is via WNT signaling which is associated with the vast numbers of different genes. Thus, in this study, we aimed to investigate whether Adenomatous Polyposis Coli gene (APC) mutation of rs41115in two locations such as 132.002 and 131.989 acts as a trigger or cause of colorectal cancer. Relatively, 30 blood samples of colorectal cancer patients and 30 normal blood samples as control group after colonoscopy and also confirmation of pathology report at Rohani Hospital in Babol (Iran) were investigated. The primers were designed in order to be included the rs41115 to identify the particular polymorphisms of gene. The polymerase chain reaction (PCR direct sequencing method) was used. Conclusively, deletion of adenine in two specific locations such as 131.989 and 132.002 has been identified, but there was no relationship between rs41115 polymorphisms located in adenomatous polyposis coli gene and colorectal cancer. Resumo: O câncer colorretal é uma das neoplasias malignas mais importantes na classificação dos cânceres gastrointestinais. Um dos fatores predisponentes no âmbito molecular para esse câncer é através da via de sinalização WNT, que está associada a um grande número de genes diferentes. Portanto, neste estudo, objetivamos investigar se a mutação rs41115 do gene da polipose adenomatosa do cólon (Adenomatous Polyposis Coli – APC) em dois locais como 132.002 e 131.989 atua como gatilho ou como causa do câncer colorretal. Relativamente, 30 amostras de sangue de pacientes com câncer colorretal e 30 amostras de sangue normal (grupo controle) foram analisadas após a colonoscopia, bem como a confirmação do laudo da patologia no Rohani Hospital em Babol (Irã). Os primers foram projetados de modo a incluir o rs41115 para identificar os polimorfismos particulares do gene. A reação em cadeia da polimerase (método de sequenciamento direto por PCR) foi utilizada. Conclusivamente, a deleção de adenina em dois locais específicos, como 131.989 e 132.002, foi identificada, mas não houve relação entre o polimorfismo rs41115 localizado no gene da polipose adenomatosa do cólon e o câncer colorretal