title: Expression and purification of human WWP2 HECT domain in Escherichia coli.
abstract: WWP2 (WW domain-containing protein 2) is an E3 ubiquitin ligase belonging to the NEDD4-like protein family involved in various cell regulations, such as carcinogenesis, transcription control and cellular transport. Compared with homologues, WWP2 is difficult to express and no practical protocols have been developed for WWP2 preparation in large scale. Recently, domain structures of homologues of WWP2 have been determined by crystallography and NMR, but none for WWP2 has been attained. In this work, through a combination of extensive screening of ∼100 constructs, expression strategies and host systems, we have found a soluble HECT domain truncation (WHP2) of WWP2 which is amendable for preparation scale expression in Escherichia coli. We have also established a relatively simple purification process to achieve highly pure WHP2 protein by employing immobilized metal-affinity chromatography followed by salting out, ion exchange chromatography and finally, size exclusion chromatography. We are able to obtain about 60mg/L of the soluble WHP2. The identity and structure of the expressed WHP2 have been analyzed by mass spectrometry and circular dichroism. The native ability of WHP2 to bind different partners has been revealed by pull-down assay.
authors.affiliation: College of Chemistry, Beijing Normal University, Beijing, China.
authors.forename: Jiahong
authors.initials: J
authors.lastname: Jiang
authors.affiliation: College of Chemistry, Beijing Normal University, Beijing, China. Electronic address: jimin_z@bnu.edu.cn.
authors.forename: Jimin
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Plant Stress Biology Center, Shanghai, China. Electronic address: ymshe@sibs.ac.cn.
authors.forename: Yimin
authors.initials: Y
authors.lastname: She
authors.affiliation: Department of Biochemical and Molecular Sciences, Queens University, Kingston, Ontario, Canada.
authors.forename: Zongchao
authors.initials: Z
authors.lastname: Jia
mesh_terms: D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D008969:Molecular Sequence Data; D051059:PTEN Phosphohydrolase / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C465283:WWP2 protein, human; D044767:Ubiquitin-Protein Ligases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D000645:Ammonium Sulfate
keywords: Escherichia coli; HECT domain expression; Human WWP2; Pull-down assay; Purification