Aqueous extraction of recombinant human proinsulin from transgenic maize endosperm

Different plant species have been used as systems to produce recombinant proteins. Maize is a crop considered to have a large potential to produce high levels of recombinant proteins and is the host for the recombinant proteins from plants currently available on the market. In the development of a plant system to produce a recombinant proteins it is important to consider the costs related to downstream processing. Also, the steps necessary to achieve the protein purity required will be highly influenced by the quality of the extract obtained. In this study, we analyzed aqueous extracts from the endosperm of transgenic maize expressing recombinant human proinsulin (rhProinsulin). A study of the effects of the variables pH and ionic strength on the extraction efficiency was carried out using experimental design and response surface methodology. Besides the concentration of the recombinant protein, the characteristics of the extracts were evaluated in terms of concentration of native components (proteins, carbohydrates, and phenolic compounds) and extract filterability. The highest rhProinsulin concentration (97.33 ng/mL) was found with a 200 mM NaCl pH 10.0 extraction solution. Under this experimental condition the concentrations of total soluble proteins, carbohydrates, and phenolics were 2.01 mg/mL, 2.21 mg/mL, and 0.11 mmol/L, respectively.2151466147

Novel Peptide Sequence (“IQ-tag”) with High Affinity for NIR Fluorochromes Allows Protein and Cell Specific Labeling for In Vivo Imaging

Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes.Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes. The developed peptide sequence ("IQ-tag") allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging.The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development

Contrast medium-induced nephropathy. Aspects on incidence, consequences, risk factors and prevention

Contrast media-induced nephropathy (CIN) is a well-known complication of radiological examinations employing iodine contrast media (I-CM). The rapid development and frequent use of coronary interventions and multi-channel detector computed tomography with concomitant administration of relatively large doses of I-CM has contributed to an increasing number of CIN cases during the last few years. Reduced renal function, especially when caused by diabetic nephropathy or renal arteriosclerosis, in combination with dehydration, congestive heart failure, hypotension, and administration of nephrotoxic drugs are risk factors for the development of CIN. When CM-based examinations cannot be replaced by other techniques in patients at risk of CIN, focus should be directed towards analysis of number and type of risk factors, adequate estimation of GFR, institution of proper preventive measures including hydration and post-procedural observation combined with surveillance of serum creatinine for 1-3 days. For the radiologist, there are several steps to consider in order to minimise the risk for CIN: use of “low-“ or “iso-osmolar” I-CM and dosing the I-CM in relation to GFR and body weight being the most important as well as utilizing radiographic techniques to keep the I-CM dose in gram iodine as low as possible below the numerical value of estimated GFR. There is as yet no pharmacological prevention that has been proven to be effective

Adsorption of glucagon and insulin on an immobilized metal ion affinity chromatography silica matrix

Glucagon is a hormone that increases blood glucose concentrations and is used as a pharmaceutical product mainly in the treatment of hypoglycaemia associated with diabetes. Given both its importance and high current cost, improved purification processes are in demand. By using immobilized metal ion affinity chromatography (IMAC), we conducted tests aimed at the purification of glucagon from complex mixtures. Adsorption studies of glucagon, insulin and a mixture of the two on adsorbents of silica-IDA-Me2+ (Cu2+, Ni2+ and Zn2+) were carried out. Fixed-bed chromatographic experiments were performed and the adsorption affinity verified for all three metals tested. The most promising condition for glucagon and insulin separation was achieved by using Ni2+ as the metal ligand and desorption with a pH step gradient. Two industrial insulin-processing fractions (one glucagon-rich and the other insulin-rich) were evaluated qualitatively under these conditions, resulting in an increase in glucagon purity for both fractions with a purification factor of five for the latter.O TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE FEVEREIRO DE 2015.211088389

Aqueous extraction of maize endosperm: Insights for recombinant protein hosts based on downstream processing

The use of transgenic plants for the production of heterologous proteins for pharmaceutical applications has increased continuously, since this provides a viable alternative to traditional expression systems. The choice of the host crop for such recombinant proteins has a significant influence on downstream processing and overall process economics. Varieties with less oil and phenolic compounds generate a less complex extract, thus facilitating recovery and purification processes for the heterologous protein. Maize is a crop which has proved its potentia for the production of high levels of recombinant proteins. In this study, the aqueous extracts of the endosperm of six non-transgenic maize varieties were analyzed for evaluation of their potential as hosts for the production of recombinant proteins. This evaluation was based on the following parameters associated with downstream processing: extract filtrability, concentration of proteins, carbohydrates and phenolics compounds and flour oil content. The study of the extraction process was carried out using factorial design and response surface methodology. The argument for the selection of a specific maize variety is presented as well as a discussion of the optimum range of conditions for the minimization of compounds, which could negatively interfere with downstream processing operations. (c) 2005 Elsevier Ltd. All rights reserved.40103327333

Transgenic Soybean Seed as Protein Expression System: Aqueous Extraction of Recombinant beta-Glucuronidase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Soybean is one of the plant species with potential to be used as seed-based bioreactor. As part of the downstream processing (DSP) of this technology, extraction is a key step, since it defines the composition of the solution from which the recombinant product will be purified. In the present work, the characteristics of soybean seeds used as a bioreactor were evaluated from a process engineering standpoint through analysis of the influence of pH and ionic strength on the extraction of recombinant beta-glucuronidase (rGUS). Concentrations of recombinant protein and native soybean compounds were analyzed and compared with similar data from extraction studies using transgenic corn seeds as bioreactor. Efficient rGUS extraction was obtained at pH of around 5.5 with no addition of salt. Soybean seed extracts had lower levels of co-extracted native compounds, than corn seed extracts, and should be considered as a potential plant bioreactor in terms of DSP.160411571167Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Downstream process engineering evaluation of transgenic soybean seeds as host for recombinant protein production

The advantages of using seeds for the production of recombinant proteins with plant-based expression system has been demonstrated by several researchers. The high productivity makes soybean a potential system for large-scale recombinant protein production. However, there is a lack of detailed engineering studies of the downstream process (DSP) of recombinant proteins produced in transgenic soybean. In this work, we evaluated the use of transgenic soybean seeds as hosts for the production of recombinant proteins from a downstream process (DSP) engineering standpoint. Recombinant beta-glucuronidase (rGUS), was used as a model for extraction and purification studies. This study showed, that even a protein with acidic pI (rGUS) can be successfully separated from native soybean proteins, which also have acidic pI. Maximum GUS specific activity (9.5 x 10(3) U/mg) with high total activity recovery (8.9 x 10(4) U/mL) was obtained using a simple extraction solution composed of 50 mmol/L citrate buffer at pH 5.25. Purification of rGUS was evaluated by a two-step chromatographic procedure - anion-exchange followed by hydrophobic interaction chromatography - which was compared to the purification of rGUS from transgenic corn and canola. Overall purification factor and activity recovery obtained were 97.3 and 110% (a value higher than 100% probably due to removal of an inhibitor). Comparison of this study with similar ones made with corn and canola seeds indicates that in terms of DSP soybean seeds can be considered a potentially viable plant system for the production of recombinant proteins. (c) 2006 Elsevier B.V. All rights reserved.32171