The effect of e6 toxic protein expression in the growth rate of three strains of e. coli

Mohamad ehsan Madadi,1,* Zahra amini bayat,2 Mehrdad hashemi,3 Neda mousavi niri,4

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

Abstract

Introduction

Cervical cancer is the fourth most common cancer worldwide, cervical cancer and its precursors are caused by various types of the human papillomavirus (hpv). the best-studied malignancy caused by hpvs is cervical cancer and it is now believed that over 99% of cervical cancers are caused by hpvs. hpv16 and 18 are thought to account for over 60% of these. among the early eight hpv proteins, e2, e5, e6, and e7 are regarded as being crucial for hpv immune escape and malignant progression. the e6 protein interacts with the p53 tumor suppressor protein, so it represents appropriate target for development of diagnostic and therapeutic tools. e. coli is most preferred system used for the production of recombinant proteins and the availability of improved genetic tools/methods are making it more valuable than ever. major challenges faced by this expression system are the expression of unusually difficult/complex proteins with rare codons or membrane and toxic proteins. the proteins expressed either in large amount or hydrophobic in nature tend to form insoluble mass. there are some strategies to improve expression of toxic proteins, such as use a more tightly regulated promoter (bl21-ai) and constitutive expression of phage t7 lysozyme (plyss) and … . in the present study, we apply three different strain of e.coli (bl21 de3, bl21 ai, de3 plyss) to achieve best one strain.

Methods

E.coli bl21 de3, bl21ai, de3 plyss strains were transformed with pet28a–e6 vector. recombinant e. coli clones were grown in terrific broth (tb) containing kanamycin (50 µg/ml) for all strains, tetracycline (12.5 µg/ml) for bl21 ai and chloramphenicol (34 µg/ml) for plyss. bacteria were induced for the expression of the e6 cloned in pet28a with 1 mm iptg at an od600 of 1. the cell mass were harvested at different time of induction and the optical density of samples were determined.

Results

Three different spices were used for expression of hpv16-e6 antigen, , by comparing the growth rate of these three strains under the same conditions, it is observed that the highest growth rate was obtained in bl21 ai strain followed by de3 plyss and then bl21 de3.

Conclusion

Toxic proteins are proteins that cause cell death or severe cultivation and maintenance defects during the growth phase when their genes were introduced into e. coli strains. the function of the expressed toxic protein may be detrimental to the proliferation and differentiation of the host cell. highest expression level of e6 in bl21 ai strain showed that tight regulation of expression using pbad promoter is the most efficient method for this protein and this is due to absence of this toxic protein in the growth phase so cells can grow and differentiate then recombinant protein was expressed after induction.

Keywords

Cervix cancer , e.coli, recombinant proteins, hpv16-e6 protein, growth rate