بهینه سازی بیان تولید پروتئین نوترکیب PapG.AcmA در ﺳﻮﯾﻪ بیانی اﺷﺮﯾﺸﯿﺎ ﮐﻠﯽ
محورهای موضوعی : میکروب شناسی مولکولیفاطمه اشرفی 1 , محمدرضا معصومیان 2 , امیر میرزایی 3
1 - مربی، گروه زیست شناسی، دانشگاه آزاد اسلامی، واحد تهران شمال، تهران
2 - کارشناس ارشد، گروه فن آوری زیستی و علوم زیستی، دانشگاه صنعتی مالک اشتر، تهران
3 - دکترا، باشگاه پژوهشگران جوان و نخبگان، دانشگاه آزاد اسلامی، واحد تهران شرق، تهران
کلید واژه: PapG.AcmA, ﺑﻬﯿﻨﻪ ﺳﺎزی بیان, اشریشیا کلی یوروپاتوژن,
چکیده مقاله :
سابقه و هدف: اشریشیا کلی یوروپاتوژن (UPEC) یکی از مهمترین باکتری های بیماری زاست که باعث عفونت مجاری ادراری می شود و هنوز هیچ واکسن انسانی علیه آن ساخته نشده است. هدف از این مطالعه، ﺑﻬﯿﻨﻪ ﺳﺎزی بیان پروتئین ﻧﻮﺗﺮﮐﯿﺐ PapG به همراه پروتئین لنگری لاکتوباسیلوس به نام AcmA در اشریشیا کلی می باشد. ﻣﻮاد و روشﻫﺎ: ژن کدکننده PapG.AcmA در وکتور کلونینگ pEXA به صورت سنتتیک خریداری و در وکتور بیانی pET21a ساب کلون گردید. ﺑﯿﺎن ﭘﺮوﺗﺌﯿﻦ نوترکیب در میزبان بیانی اشریشیا کلی سویه اریگامی با روش SDS-PAGE و وسترن بلات مطالعه شد. تولید پروتئین فیوژن در مقیاس بالا تحت تاثیر ﺗﺮﮐﯿﺐ ﻣﺤﯿﻂ ﮐﺸﺖ، ﻏﻠﻈﺖﻫﺎی ﻣﺨﺘﻠﻒ IPTG به ﻋﻨﻮان اﻟﻘﺎﮔﺮ و زﻣﺎن اﻟﻘﺎ در ﺑﯿﺎن پروتئین ﻧﻮﺗﺮﮐﯿﺐ PapG.AcmA بهینهﺳﺎزی گردید. ﯾﺎﻓﺘﻪﻫﺎ: با توجه به نتایج ﺑﻬﺘﺮﯾﻦ بیان در مقیاس بالا، ﺗﻮﺳﻂ ﻏﻠﻈﺖ 0.1 میلی مولار IPTG در ﮐﺪورت ﻧﻮری OD600nm برابر با 3 تعیین ﺷﺪ. ﻣﺤﯿﻂ ﮐﺸﺖ ﭘﯿﭽﯿﺪه اﺻﻼح ﺷﺪه ﺣﺎوی گلوکز (6 گرم بر لیتر)، عصاره مخمر (20 گرم بر لیتر)، تریپتون (10 گرم بر لیتر)، KH2PO4 (2.3 گرم بر لیتر) و K2HPO4 (12.5 گرم بر لیتر) ﺑﻪ ﻋﻨﻮان ﻣﺤﯿﻂ ﮐﺸﺖ ﺑﻬﯿﻨﻪ ﺗﻌﯿﯿﻦ ﮔﺮدﯾﺪ. ﺑﯿﺸﺘﺮﯾﻦ ﻣﻘﺪار ﺗﻮﻟﯿﺪ ﺑﯿﻮﻣﺲ و ﺑﯿﺎن ﭘﺮوﺗﺌﯿﻦ ﻫﺪف در ﻣﺤﯿﻂ ﮐﺸﺖ ﺑﻬﯿﻨﻪﺳﺎزی ﺷﺪه ﺑﻪ ﻣﯿﺰان OD600nm برابر با 5.5 ﺗﻌﯿﯿﻦ شد. ﻧﺘﯿﺠﻪﮔﯿﺮی: نتایج نشان داد که کلون سازی و بیان ژن PapG.AcmA قابل انجام می باشد. همچنین اﺳﺘﻔﺎده از ﻣﻨﺒﻊ ﮐﺮﺑﻦ ﮔﻠﻮﮐﺰ در ﺗﺮﮐﯿﺐ ﻣﺤﯿﻂ ﮐﺸﺖ ﭘﯿﭽﯿﺪه ﻧﺴﺒﺖ ﺑﻪ ﻣﺤﯿﻂ ﮐﺸﺖ LB بیان ﺑﻬﺘﺮی ﻣﯽدﻫﺪ. در نهایت با تخلیص و ارزیابی ایمنی زایی پروتئین نوترکیب، می توان از آن به عنوان یک کاندیدای واکسن استفاده نمود.
Background & Objectives: Uropathogenic Escherichia coli (UPEC) is one of the most common bacteria to cause urinary tract infection (UTI). No human vaccine against UTI has yet been developed. The aim of this study was to optimize the expression of recombinant PapG with Lactobacillus anchor protein AcmA in E. coli. Materials & Methods: The synthetic cloning vector, pEXA containing PapG.AcmA was purchased and subcloned into pET21a vector. The protein expression levels in Origami expression host (E. coli) were analyzed by SDS-PAGE gel and western blotting. Moreover, various concentrations of IPTG (Isopropyl Thiogalactopyranoside), the medium component and induction time was optimized for large scale expression of recombinant protein. Results: Based on results, optimum expression in large scale was occurred in 0.1mM IPTG and OD= 3 optical density. The modified complex culture medium containing: glucose 6 g/I, K2HPO4 12.5 g/l, KH2PO4 2.3 g/l, Yeast Extract 20 g/l, tryptone 10 g/l were determined as optimal medium. OD 600nm= 3.0 was determined as the best time for induction by IPTG at a concentration of 0.1 mM. The levels of the expression of the target protein was determined at OD600nm= 5.5. Conclusion: Based on the result, we were able to do cloning and expression of PapG.AcmA. Addition of extra carbon source (glucose) to the complex medium caused a better PapG.AcmA recombinant protein expression. Finally, by purification of recombinant protein and evaluation of its immunogenicity, it can be used as a vaccine candidate against the urinary tract infection.
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11. Song D, Gu Q. Surface expression of Helicobacter pylori urease subunit B gene E fragment on Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A. Biotechnol Lett. 2009; 31(7): 985-989.
12. Swartz JR. Advances in Esherichia coli production of therapeutic proteins. Curr Opin Biotechnol. 2001; 12: 195-201.
13. Varedi Koolaee SM, Shojaosadati SA, Babaeipour V, Ghaemi N. Physiological and morphological changes of recombinant E. coli during over-expression of human interferon-g in HCDC. Iran J Biotech. 2006; 4: 230-238.
14. Collins T, Azevedo-silva J, Costa A, Branca F, Machado R, Casal M. Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimization. Microb Cell Fact. 2013; 1: 12-21.
15. Chong M, Leung R, Wong C, Yuen A. The effects of ampicillin versus tetracycline on the plasmid. Microbial Immunol. 2003; 3: 87-95.
16. Zeinoddini M. Theoretical and practical guide to protein analysis methods.1st.Tehran Malek Ashtar University of Technology press, 2011; pp: 1-30.
17. Fong BA, Wood DW. Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation. Microbe cell Fact. 2010; 77: 9-16.
18. Vélez AM, da Silva AJ, Luperni Horta AC, Sargo CR, Campani G, Gonçalves Silva G, de Lima Camargo Giordano R, Zangirolami TC. High-throughput strategies for penicillin G acylase production in r E. coli Fed-batch cultivations. BMC Biotechnol. 2014; 21: 6-14.
19. Muntari B, Amid A, Mel M, Jami MS, Salleh HM. Recombinant bromelain production in Escherichia coli. AMB Express. 2012; 2: 12.
20. Kelley KD, Olive LQ, Hadziselimovic A, Sanders CR. Look and see if time to induce protein expression in Escherichia coli cultures. Biochem. 2010; 49: 5405-5460.
21. Lecina M, Sarro E, Casablancas A, Godia F, Caire J. IPTG limitation avoids metabolic burden and acetic acid accumulation in induced fed-batch cultures of Escherichia coli M15 under glucose limiting conditions. Biochem Eng J. 2013; 70: 78-83.
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1. Hooton TM. Recurrent urinary tract infection in women. Int J Antimicrob Agents. 2001; 17(4): 259-268.
2. Johnson JR, Owens K, Gajewski A, Kuskowski MA. Bacterial characteristics in relation to clinical source of Escherichia coli isolates from women with acute cystitis or pyelonephritis and uninfected women. J Clin Microbiol. 2005; 43(12): 6064-6072.
3. Dodson KW, Pinkner JS, Rose T, Magnusson G, Hultgren SJ, Waksman G. Structural basis of the interaction of the pyelonephritic E. coli adhesion to its human kidney receptor. Cell. 2001; 105(6): 733-743.
4. Yasmeen Kausar SKC, Nadagir SD, Halesh LH, Chandrasekhar MR. Virulence factors, serotypes and antimicrobial susceptibility pattern of Escherichia coli in urinary tract infections. J Med Sci. 2009; 2(1): 47-55.
5. Snyder JA, Lloyd AL, Lockatell CV, Johnson DE, Mobley HL. Role of phase variation of type 1 fimbriae in a uropathogenic Escherichia coli cystitis isolate during urinary tract infection. Infect Immun. 2006; 74(2): 1387-1393.
6. Lane MC, Mobley HLT. Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney. Kidney Int. 2007; 72(1): 19-25.
7. Al-Mayahie SMG. Vaginal colonization by papG allele II(+) Escherichia coli isolates from pregnant and non-pregnant women as predisposing factor to pyelonephritis. Infect Dis Obstetrics Gynecol. 2013; Article ID 860402.
8. Roberts JA, Kaack MB, Baskin G, Chapman MR, Hunstad DA, Pinkner JS. Antibody responses and protection from pyelonephritis following vaccination with purified Escherichia coli PapDG protein. J Urol. 2004; 171(4): 1682-1685.
9. Liu MA. DNA vaccines: a review. J Intern Med. 2003; 253(4): 402-410.
10. Raha AR, Varma NRS, Yusoff K, Ross E, Foo HL. Cell surface display system for Lactococcus lactis: a novel development for oral vaccine. Appl Microbiol Biotechnol. 2005; 68(1): 75-81.
11. Song D, Gu Q. Surface expression of Helicobacter pylori urease subunit B gene E fragment on Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A. Biotechnol Lett. 2009; 31(7): 985-989.
12. Swartz JR. Advances in Esherichia coli production of therapeutic proteins. Curr Opin Biotechnol. 2001; 12: 195-201.
13. Varedi Koolaee SM, Shojaosadati SA, Babaeipour V, Ghaemi N. Physiological and morphological changes of recombinant E. coli during over-expression of human interferon-g in HCDC. Iran J Biotech. 2006; 4: 230-238.
14. Collins T, Azevedo-silva J, Costa A, Branca F, Machado R, Casal M. Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimization. Microb Cell Fact. 2013; 1: 12-21.
15. Chong M, Leung R, Wong C, Yuen A. The effects of ampicillin versus tetracycline on the plasmid. Microbial Immunol. 2003; 3: 87-95.
16. Zeinoddini M. Theoretical and practical guide to protein analysis methods.1st.Tehran Malek Ashtar University of Technology press, 2011; pp: 1-30.
17. Fong BA, Wood DW. Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation. Microbe cell Fact. 2010; 77: 9-16.
18. Vélez AM, da Silva AJ, Luperni Horta AC, Sargo CR, Campani G, Gonçalves Silva G, de Lima Camargo Giordano R, Zangirolami TC. High-throughput strategies for penicillin G acylase production in r E. coli Fed-batch cultivations. BMC Biotechnol. 2014; 21: 6-14.
19. Muntari B, Amid A, Mel M, Jami MS, Salleh HM. Recombinant bromelain production in Escherichia coli. AMB Express. 2012; 2: 12.
20. Kelley KD, Olive LQ, Hadziselimovic A, Sanders CR. Look and see if time to induce protein expression in Escherichia coli cultures. Biochem. 2010; 49: 5405-5460.
21. Lecina M, Sarro E, Casablancas A, Godia F, Caire J. IPTG limitation avoids metabolic burden and acetic acid accumulation in induced fed-batch cultures of Escherichia coli M15 under glucose limiting conditions. Biochem Eng J. 2013; 70: 78-83.
