| Summary: | Recombinant proteins can be produced in bacteria, yeast, insect cells, mammalian cells, and cell-free system. Recombinant proteins are expressed as inclusion bodies (IBs) in bacterial enriched native-like secondary structure and thus give a great potential in biotechnological utilities. IBs are produced in Escherichia coli cells and solubilization process is required to recover desired protein in bioactive form. In this study, the effects of solubilization methods on the recovery of soluble enhanced green fluorescent protein (EGFP) from IBs by using urea, alkyl alcohol and freeze thaw method were investigated. The present study indicates urea concentration, incubation temperature, type of alcohol and its concentration, freezing duration and freeze thaw cycles influenced the yield and purity of solubilized EGFP. Conventional method using 8 M of urea with incubation temperature of 60°C achieved the highest yield (61%) and purity (10%). Mild IBs solubilization with 6 M of n-butanol and 2 M of urea has solubilized IBs with a yield of 45% and purity of 22%. By freezing and thawing the IBs suspension in 2 M of urea, the yield (66%) and purity (9%) of solubilized EGFP were comparable to that of 8 M of urea in buffer. Hence, mild solubilization using the alkyl alcohol or freeze thaw method is applicable for IBs solubilization. Previous studies reported the quality and nativity of refolded soluble protein from inclusion body is questionable because the refolded protein with wrong conformation will assemble to form soluble aggregates. Many studies involving proteins from inclusion bodies only assessed the protein quality based on the protein solubility and functionality, but not the protein conformation that reflects the protein aggregation tendency. In this study, EGFP-IBs was used as the model protein to investigate the soluble aggregates formation under different solubilization and refolding conditions. The present study used a gel-based imaging method to analyze the refolded soluble protein based on fluorescence intensity, charge, shape, and size of the protein. For the solubilized inclusion bodies refolding under high protein concentration and low protein conditions, aggregation can be visualized with polyacrylamide gels. Gel images showed the refolded soluble protein changed in conformation and increased in size when the solubilized inclusion bodies underwent various refolding periods. Meanwhile, the refolded soluble protein under the refolding condition of low protein concentration and high protein purity has a correct protein conformation and achieved the highest refolding yield. Studying the effects of refolding conditions using different types of solubilized inclusion bodies may provide researchers with possible approaches to avoid soluble aggregates formation in the pharmaceutical and nanobiotechnology applications. By using PNU-PAGE for clarifying and purifying the solubilized EGFP prior to refolding process, the method has successfully recovered 2.4 μg of folded soluble EGFP with 12.4% of refolding yield and 52.2% of purity after one day of refolding incubation period.
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