Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage
| Autor: | Ariane Leites Larentis, Ellen Jessouron, Ricardo Galler, Marco Alberto Medeiros, Ana Paula Corrêa Argondizzo, Gabriela dos Santos Esteves |
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| Rok vydání: | 2010 |
| Předmět: |
Isopropyl Thiogalactoside
Antigenicity Drug Storage Lipoproteins Blotting Western lac operon Protein degradation Biology medicine.disease_cause law.invention Plasmid Western blot law medicine Escherichia coli Cloning Molecular Adhesins Bacterial medicine.diagnostic_test Protein Stability Temperature Reproducibility of Results Chromatography Ion Exchange Molecular biology Recombinant Proteins Bacterial adhesin Glucose Biochemistry Recombinant DNA Electrophoresis Polyacrylamide Gel Biotechnology |
| Zdroj: | Protein expression and purification. 78(1) |
| ISSN: | 1096-0279 |
| Popis: | The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage. |
| Databáze: | OpenAIRE |
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