بهینه نمودن بیان و تخلیص پروتئین نوترکیب لیپوپروتئین مرتبط با پپتیدوگلیکان لژیونلا پنوموفیلا

Title

Optimization of Ggene Expression and Purification of Legionella Pneumophila Peptidoglycan Associated Lipoprotein (PAL) Recombinant Protein

English Abstract

Background & Objectives: Legionellae including Legionella pneumophila, are recognized as the causative agents for community, travel and hospital-acquired pneumonia. There are many problems with most of available diagnostic tests used to diagnose Legionella pneumonia, including inadequate sensitivity and specificity, and inability to provide a result in a clinically useful time period but among them, the urinary antigen tests have revolutionaized the laboratory diagnosis of Legionella pneumonia. Since the peptidoglycan-associated lipoprotein (PAL) protein of L. pneumophila is an urinary antigen and considered as useful diagnostic antigen to diagnose Legionella infection, the aim of this study was to optimaize expression and purification of L. pneumophila PAL protein. Methods: In this experimental study, optimizing of 5 parameters (cell density, induction time, growth temperature, IPTG concentration and type of medium) was performed. After expression, periplasmic extract was prepared and recombinant PAL protein purified using Ni2+-charged resin column. Finally, recombinant PAL protein was verified by Western blotting. Results: The optimum expression of the r-PAL protein was occurred at an OD600 of 0.6, 1mM IPTG concentration, after 15 hours at 25ºC and use of Terrific Broth medium. Recombinant periplasmic PAL protein was highly purified (>80%) using Ni-NTA column. Also, western blotting analysis showed that recombinant PAL protein was specifically recognized by anti-His6-peroxidase antibody. Conclusion: In order to increase the functional expression of recombinant proteins, a number of strategies were employed such as optimization of expression conditions and periplasmic expression. Also, the results obtained from this study supported the idea that E. coli BL21 strain can be served as a functional host for pET26b vector and the designed host and vector can be used in large scale production of recombinant PAL protein

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