An improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies.

Amyloid-beta (Aß) peptide mediates several neurodegenerative diseases. The 42 amino acid (Aß1-42) is the predominant form of peptide found in the neuritic plaques and has been demonstrated to be neurotoxic in vivo and in vitro. The availability of large quantities of Aß peptide will help in several biochemical and biophysical studies that may help in exploring the aggregation mechanism and toxicity of Aß peptide. We report a convenient and economical method to obtain such a peptide biologically. Synthetic oligonucleotides encoding Aß1-42 were constructed and amplified through the polymerase cycling assembly (also known as assembly PCR), followed by the amplification PCR. Aß1-42 gene was cloned into pET41a(+) vector for expression. Interestingly, the addition of 3% (v/v) ethanol to the culture medium resulted in the production of large amounts of soluble Aß fusion protein. The Aß fusion protein was subjected to a Ni-NTA affinity chromatography followed by enterokinase digestion, and the Aß peptide was purified using glutathione Sepharose affinity chromatography. The peptide yield was ∼15mg/L culture, indicating the utility of this method for high-yield production of soluble Aß peptide. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and immunoblotting with anti-His antibody confirmed the identity of purified Aß fusion protein and Aß peptide. In addition, this method provides an advantage over the chemical synthesis and other conventional methods used for large-scale production of recombinant Aß peptide.

Functional classification and biochemical characterization of a novel rho class glutathione S-transferase in Synechocystis PCC 6803.

We report a novel class of glutathione S-transferase (GST) from the model cyanobacterium Synechocystis PCC 6803 (sll1545) which catalyzes the detoxification of the water pollutant dichloroacetate and also shows strong glutathione-dependent peroxidase activity representing the classical activities of zeta and theta/alpha class respectively. Interestingly, sll1545 has very low sequence and structural similarity with these classes. This is the first report of dichloroacetate degradation activity by any bacterial GST. Based on these results we classify sll1545 to a novel GST class, rho. The present data also indicate potential biotechnological and industrial applications of cyanobacterial GST in dichloroacetate-polluted areas.

Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae.

We report the molecular cloning, expression, and single-step homogeneous purification of RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae. RPB5 is a 210 amino acid nuclear protein that functions as the fifth largest subunit of polymerase II and plays a central role in transcription. The gene that codes for RPB5 was generated by amplification by polymerase chain reaction. It was then inserted in the expression vector pET28a(+) under the transcriptional control of the bacteriophage T7 promoter and lac operator. BL21(DE3) Escherichia coli strain transformed with the rpb5 expression vector pET28a(+)-rpb5 accumulates large amounts of a soluble protein of about 30 kDa (25 kDa plus 5 kDa double His6-Tag at N and C-terminal). The protein was purified to homogeneity using immobilized metal affinity chromatography. RPB5 recombinant protein was further confirmed by immunoblotting with anti-His antibody. In this study, the expression and purification procedures have provided a simple and efficient method to obtain pure RPB5 in large quantities. This will provide an opportunity to study the role of S. cerevisiae RPB5 in gene expression and transcription regulation. Furthermore, it can provide additional knowledge of the interaction partners of RPB5 during various steps of transcription and gene expression.