The study of the level of recombinant e7 expression in four strains of e.coli
,1,* Zahra amini bayat
,2 Neda mousavi niri
,3 Mehrdad hashemi
1. . Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, IR Iran
2. Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
3. Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, IR Iran
4. Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, IR Iran
Cervical cancer is one of the leading world causes of cancer morbidity and mortality in woman, with more than 98% related to a human papillomavirus (hpv) infection origin. infection with specific subtypes of hpv has been strongly implicated in cervical carcinogenesis. the identification and functional verification of host proteins associated with hpv e6 and e7 oncoproteins may provide useful information in understanding cervical carcinogenesis and the development of cervical cancer-specific markers. the advent of functional genomics and proteomics has provided hope of discovering novel biological markers for use in the screening, early diagnosis, prognostication and prediction of response to therapy.
infection with human papillomavirus type 16 (hpv-16) is the main factor associated with development of cervical cancer. the hpv-16 e6 and e7 proteins are constitutively produced in cervical carcinomas, and e7 was shown to interact with several cell compounds, causing deregulation of the cell cycle and cell transformation. e7 is a 98-amino-acid nuclear phosphoprotein that is devoid of any known enzymatic activity. e7 protein is widely studied because of its implication in carcinoma onset. it is also considered to be a good antigen candidate for the development of new vaccines against cervical cancer. in this study, we compared expression level of hpv e7 protein in four different strains of escherichia coli (rosetta, bl21 de3 ai, bl21 de3 plyss, bl21 de3).
for construction of a vector expressing the e7 multi-epitope protein, the synthetic coding sequence of e7 protein was cloned into pet28a(+) vector. in the recombinant vector was transformed into the bl21(de3), plyss, rosetta, bl21 (de3) ai e.coli strains then isolated colonies were grown in tb medium containing appropriate antibiotic ( kanamycin (50 µg/ml) , chloramphenicol (34 µg/ml) , tetracycline (12.5 µg/ml) and incubated in a shaker at 37°c until the optical density at 600 nm (od600) reached 1. protein expression was induced by adding 1 mm isopropyl-β thiogalactopyranoside (iptg) and the cultures were incubated at 37°c for 4 h or overnight. cells were harvested by centrifugation (22 o c, 6000rpm, 5 minute) and lysed by lysis buffer (urea 8m) and sonication. the recombinant protein was characterized by sds-page
for the best expression of hpv-16e7, four expressing e.coli strains (de3) the expression patterns of the recombinant protein were analyzed.e7 protein was expressed in all four strain of e.coli in the same condition. the best expression level was achieved in rosetta strain after 4 hour induction following overnight induction.18 % of total protein obtained in rosetta strain compared to 10% in bl21(de3).
Cervical cancer is a worldwide public health problem among women, especially in emerging nations. to improve the control of cervical cancer, new adjuvant diagnostic and therapeutic strategies are required. advances in immunology, genomics and proteomics have accelerated our understanding of the genetic and cellular basis of many cancer types. cervical cancer is a member of the virus-related neoplasms, with its initiation and promotion associated with persistent infection of oncogenic hpv.
rosetta host strains are bl21 lazy derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in e.coli. these strains supply trnas for the codons aua, agg, aga, cua, ccc, gga on a compatible chloramphenicol resistant plasmid. because of the presence of some rare codon in the e7 gene sequence cloned in the vector, using rosetta strain overcome this problem and the better yield was achieved in this strain.
E.coli, recombinant proteins, hpv-e7 protein, expression level , cervix cancer