Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture medium and in vitro refolding to increase recombinant proteins production. Additives increase protein refolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process. In this work, a comparative study was performed on refolding of recombinant plasminogen activator (rPA) in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA inclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilution method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride (Gn-HCl) and Dithiothreitol (DTT) on refolding process were investigated. The refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10. In refolding step, Larginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM. Glutathione pairs (GSH-GssG) showed refolding enhancer effect in a range of 2-20 mM. The highest refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10:1 ratio in pH 10. In conclusion, the results show that L-arginine plays an important role in the refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct refolding. The results also revealed that higher solubility in the pres

http://www.ijbiotech.com/article_7140_a7651af40f069fcfee468002418e0520.pdf

Optimizing refolding condition for recombinant tissue plasminogen activator

Aim: Angiogenesis plays a critical role in tumour formation and metastasis. Suitable radiolabeled angiogenesis inhibitor can be used for noninvasive imaging of angiogenesis and radionuclide therapy. Here we prepare rhenium-188 labeled recombinant human plasminogen kringle5 (188Re-rhk5) in a convenient manner than evaluate its properties in A549 lung adenocarcinoma. Methods: 188Re-rhk5 was obtained by conjugating His group at the C end of rhk5 with fac-[188Re(H2O)3(CO)3]+. Chelating efficiency of fac-[188Re(H2O)3(CO)3]+ and radiolabeling efficiency of 188Re-rhk5 were measured by radio thin-layer chromatography (RTLC). In vitro stability of 188Re-rhk5 was determined in human serum at 37°C and analyzed by RTLC. Competition test was also performed to verify the specificity of binding. A biodistribution study was carried out in nude mice bearing A549 lung adenocarcinoma. Results: 188Re-rhk5 was obtained with a radiolabel efficiency of 66.1%, the radiochemical purity (RCP) can reach 95.2% after purification. 188Re-rhk5 showed high stability in human serum, the RCP was more than 80% even 12 h after incubation. Competition test showed a high binding specificity. Furthermore, this radio-complex was excreted mainly through kidneys and showed specific tumour uptake in mice bearing A549 tumours. Conclusion: 188Re-rhk5 was prepared by a simple method. Preliminary biodistribution results showed its potential as an agent for possible tumour imaging, therapy and encouraged further investigation.

Rhenium-188 labeled recombinant human plasminogen kringle5 (rhk5) and preliminary biodistribution. Evaluation in mice bearing A549 tumours.

Targeting drugs to receptors involved in tumor angiogenesis is considered to be a novel and promising approach to improve cancer treatment. This study aimed to evaluate the anti-tumor efficacy of 188Re-labeled recombinant human plasminogen kringle 5 (188Re‑rhk5) through [18F]-fluorodeoxyglucose (FDG) micro-positron emission tomography (PET). Radiolabeled rhk5 was obtained by conjugating the hystidine (6 x His) group at the carbon end of rhk5 with fac-[188Re(H2O)3(CO)3]+. The biodistribution study of 188Re-rhk5 showed that 188Re-rhk5 had a high initial tumor uptake and prolonged tumor retention. The highest tumor uptake of 188Re-rhk5 (3.65±0.82% ID/g) was found 2 h after injection which decreased to 0.81±0.14% ID/g 12 h after injection. Following therapy, tumor size measurement indicated that 188Re-rhk5-treated tumors were smaller than 188Re-, rhk5- and saline-treated controls 6 days after the treatment. In vivo 18F-FDG micro-PET imaging showed significantly reduced tumor metabolism in the 188Re-rhk5-treated mice vs. those treated with rhk5, 188Re and saline control, 1 day after treatment. Moreover, the number of microvessels was significantly reduced after 188Re-rhk5 treatment as determined by CD31 staining. Our results demonstrate that specific delivery of 188Re-rhk5 allows preferential cytotoxicity to A549 lung cancer cells and tumor vasculature. 18F-FDG micro-PET is a non-invasive imaging tool that can be utilized to assess early tumor responses to 188Re-rhk5 therapy.

/pdf/evaluation-of-biodistribution-and-antitumor-effects-of-188re-dwal3q8lu4.pdf

Evaluation of biodistribution and antitumor effects of 188Re-rhk5 in a mouse model of lung cancer

Cloning, expression and purification of kringle 1 domaln of human plasminogen in escherichia coli

Rhenium-188 markiertes, rekombinantes humanes Plasminogen Kringle 5 (rhk5) und vorläufige Bioverteilung – Beurteilung bei Mäusen mit A549-Tumoren

The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelial cell specifically, and shows promise in anti‐angiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When previously expressed in Escherichia coli, recombinant kringle 5 mainly deposited as inactive, insoluble inclusion bodies and the refolding yield was low. In the present study, human kringle 5 was fusion‐expressed with GST (gluthathione‐S‐transferase) under the control of T7 promoter in E. coli. The IPTG‐induced GST‐kringle 5 was about 20% of the total cellular proteins and, among the expressed GST‐kringle 5 proteins, 80% was present in the supernatant. The GST‐kringle 5 fusion protein exhibited some anti‐proliferation activity towards bovine capillary endothelial cells. After GST‐kringle 5 purification, subsequent enterokinase release of intact kringle 5 from the ...

Expression of Biologically Active Kringle 5 Domain of Human Plasminogen in Escherichia coli

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Expression of Biologically Active Kringle 5 Domain of Human Plasminogen in Escherichia coli