title: Simplified method to obtain enhanced expression of tau protein from E. coli and one-step purification by direct boiling.
abstract: Tau is an intrinsically disordered protein responsible for maintaining the structure and stability of axonal microtubules. However, in certain disease conditions including Alzheimer's disease, tau protein may undergo biochemical and structural changes to form intracellular aggregates. Since tau is a proline- and arginine-rich eukaryotic protein, heterologous expression in Escherichia coli often results in poor yield and has been a major technical challenge. In the current work, we have improved the expressed yield of tau by overcoming codon bias problem and established a simplified protocol for efficient extraction. The reported method has two distinct features: (i) enhanced tau expression (upto eightfold) by supplementing deficient tRNAs that aid in rapid translation and (ii) direct boiling of expressed E. coli cells to extract tau with no separate cell lysis step. We further demonstrate that tau extracted by the direct boiling method is similar to tau purified by size-exclusion chromatography exhibiting similar structural and biophysical characteristics including aggregation propensity. Since morphologies and in vitro toxicity of fibrillar tau aggregates were also similar, tau extracted by the one-step direct boiling method can be used for tau aggregation assays without any additional purification.
authors.affiliation: a Biological Engineering , Indian Institute of Technology Gandhinagar, Palaj , Gandhinagar , India.
authors.forename: V Guru
authors.initials: VG
authors.lastname: KrishnaKumar
authors.affiliation: a Biological Engineering , Indian Institute of Technology Gandhinagar, Palaj , Gandhinagar , India.
authors.forename: Sharad
authors.initials: S
authors.lastname: Gupta
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics*; D005615:Freezing; D015870:Gene Expression; D006358:Hot Temperature; D006801:Humans; D066329:Protein Aggregates; D012343:RNA, Transfer / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D013010:Sonication; D015854:Up-Regulation; D016875:tau Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification* / Q000648:ultrastructure
chemical_list: D066329:Protein Aggregates; D011994:Recombinant Proteins; D016875:tau Proteins; D012343:RNA, Transfer
keywords: Aggregation assay; Alzhemier’s disease; codon bias; direct boiling; protein expression; tau