title: Incorporation of a tag helps to overcome expression variability in a recombinant host.
abstract: Epigenetics have witnessed a renewed interest over the past decade and assays with recombinant histones has become an important tool for uncovering various aspects of histone biology. However, at times absence of recombinant histone accumulation in bacteria is encountered which is also commonly observed for many eukaryotic proteins in general. In this study, we have investigated the effect of multiple parameters on heterologous expression of proteins. We show that there is marked variability in the accumulation of H2A.2, H2B.1, H3.2 and H4 in the recombinant host, possibly owing to translational variability and degradation by the host proteases. We found that the variability could be overcome by incorporation of the commonly used purification tags, like GST or MBP, of appropriate size and position. Our results provide compelling evidence that transcript parameters like rare codon and GC content, mRNA secondary structure etc. together modulate translation kinetics and govern recombinant protein accumulation.
authors.affiliation: Epigenetics and Chromatin Biology Group, Gupta Lab, India.
authors.forename: Saikat
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: Epigenetics and Chromatin Biology Group, Gupta Lab, India.
authors.forename: Divya
authors.initials: D
authors.lastname: Reddy
authors.affiliation: Integrated Biophysics and Structural Biology Lab, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, MH, India.
authors.forename: Raja
authors.initials: R
authors.lastname: Reddy
authors.affiliation: Epigenetics and Chromatin Biology Group, Gupta Lab, India.
authors.forename: Asmita
authors.initials: A
authors.lastname: Sharda
authors.affiliation: Integrated Biophysics and Structural Biology Lab, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, MH, India.
authors.forename: Kakoli
authors.initials: K
authors.lastname: Bose
authors.affiliation: Epigenetics and Chromatin Biology Group, Gupta Lab, India.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Gupta
keywords: CAI, codon adaptation indexes; DUSP1, dual specificity phosphatase 1; GAPDH, glyceraldehyde phosphate dehydrogenase; GST, glutathione-S-transferase; HAX-1, human protein HCLS-1 associated protein X-1; Histones; IPTG, Isopropyl β-d-1-thiogalactopyranoside; MALDI, matrix-assisted laser desorption/ionization; MBP, maltose binding protein; Misfolding; NAP1, nucleosome assemble protein 1; PP1, protein phosphatase 1; RBS, ribosome-binding site; RT-PCR, reverse transcriptase polymerase chain reaction; Rare codons; TMAO, trimethylamine oxide; Translation; Truncated GST