title: E6 protein of human papillomavirus 16 (HPV16) expressed in Escherichia coli sans a stretch of hydrophobic amino acids, enables purification of GST-ΔE6 in the soluble form and retains the binding ability to p53.
abstract: Recombinant E6 expressed in Escherichia coli is known to form recalcitrant inclusion bodies even when fused to the soluble GST protein. This study describes the modification of the HPV genotype-16 oncogenic protein E6 in order to obtain it in the soluble form. The modified protein (ΔE6) was expressed in E. coli BL21 as an N-terminal fusion with GST (GST-ΔE6). ΔE6 was constructed by deleting the nucleotide sequences coding for IHDIIL (31-36 a.a), one of the highly hydrophobic peptide stretches, using splicing by overextension polymerase chain reaction (SOE-PCR). The removal of IHDIIL residues rendered the GST-ΔE6 soluble and amenable for purification involving a two step process a preliminary glutathione-GST affinity chromatography followed by gel-filtration chromatography. Evaluation of purified protein fractions by HPLC suggests that GST-ΔE6 exists as a monomer. Further, the ΔE6 in GST-ΔE6 seemed to retain the binding ability to p53 as determined by the glutathione-GST capture ELISA. Purified GST-ΔE6 we reckon, might find use as an essential reagent in immunological assays, in sero-epidemiological studies, and also in studies to delineate the structure and function of HPV16 E6.
authors.affiliation: R&D Centre, Indian Immunologicals Ltd., Rakshapuram, Gachibowli P.O., Hyderabad 500032, India.
authors.forename: Ravi Ranjan
authors.initials: RR
authors.lastname: Verma
authors.forename: Rajan
authors.initials: R
authors.lastname: Sriraman
authors.forename: Samir Kumar
authors.initials: SK
authors.lastname: Rana
authors.forename: N M
authors.initials: NM
authors.lastname: Ponnanna
authors.forename: Burki
authors.initials: B
authors.lastname: Rajendar
authors.forename: Priyanka
authors.initials: P
authors.lastname: Ghantasala
authors.forename: Lingala
authors.initials: L
authors.lastname: Rajendra
authors.forename: Ramesh V
authors.initials: RV
authors.lastname: Matur
authors.forename: Villupanoor Alwar
authors.initials: VA
authors.lastname: Srinivasan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030361:Papillomavirus Infections / Q000821:virology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53
keywords: GST-ΔE6; HPV16 E6; Oligomeric state; ProtScale and ProtParam analysis; SOE-PCR