Buffer exchange was followed by diafiltration into 10 mM Tris pH 7.4 using a 10,000 MWCO centrifugal filter. 24 h before analysis by CCK-8 assay, absorbance at 450 nm. Results are expressed as mean s.e.m following conversion to % viability. ANOVA with Dunnett’s post-hoc analysis was performed (*p 0.05), n = 6 for live and dead controls and n = 3 for LC incubated cells.(TIF) pone.0206167.s003.tif (49K) GUID:?306B8060-4707-4AD6-8593-9D82BEA4775C S3 Fig: Subcellular localisation of internalised FITC conjugated LEN assessed by analysis of Z-stack. Optical sectioning of complete z-stacks reveals FITC-labelled LEN (green) is usually on the same focal plane as the cell nucleus (HoechstCblue) indicating the VL is usually inside the cells and not surface bound. Top right panel shows enlarged image of z-slice 13 marked asterisks.(TIF) pone.0206167.s004.tif (505K) GUID:?A825628A-09D7-4A3C-B33C-A9D2CE0E93BC S4 Fig: Subcellular localisation of internalised FITC conjugated SMA assessed by analysis of Z-stack. Optical sectioning of complete Z-stacks reveals FITC-labelled SMA (green) is usually on the same focal plane as the nuclei (HoechstCblue) indicating the VL is usually inside the cells and not surface bound. Top right panel shows enlarged image of z-slice 13 marked asterisks.(TIF) pone.0206167.s005.tif (809K) GUID:?EB4BFC51-BB4A-4C13-BC4F-DA9AC051E493 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Immunoglobulin light chain amyloidosis is SCH 23390 HCl the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is usually amyloidogenic characterization of these proteins revealed that SMA is usually significantly less stable than LEN [7, 10, 11], displaying enhanced fibrillation kinetics. Such dramatic differences SCH 23390 HCl arise only from a few amino acid substitutions, where SMA is usually altered by 8 residues (S29N, K30R, P40L, Q89H, T94H, Y96Q, S97T and I106L). the amyloid potential of these proteins as well as many other VLs and the effect on cells/mechanisms of toxicity has not been realised. A recombinant protein expression system was previously established for these VL proteins, employing lysosyme cell disruption and purification using a multi-step chromatographic strategy of strong anion and cation exchange followed by size exclusion chromatography [7]. For LEN, yields were reported to be SCH 23390 HCl around 10 mg/L, while SMA was reported to be less. B leader peptide for 6 proteins [15, 16]. There is an increasing Rabbit Polyclonal to EPHA7 (phospho-Tyr791) interest in recombinant expression of human immunoglobulin chain fragments for therapeutic use therefore improving expression yields and reproducibility of these systems are important factors to consider when designing expression strategies. Periplasmic expression offers a solution to many issues that arise when expressing proteins that are prone to mis-folding or aggregation. There are a range of different leader sequences that can and have been employed to direct light chain proteins and fragments to the periplasmic space e.g. amicyanin [17]. In the present work, we present a strategy exploiting periplasmic expression of VLs from the two light chain proteins SMA and LEN that improve on previous methods [7, 18]. Periplasmic expression can result in suboptimal yields and incomplete removal of peptide leader sequences [12]. We show that in our system we have improved yields, comparable with those obtained through the more complex refolding process and have complete SCH 23390 HCl removal of the leader sequence confirmed by mass spectrometry. We pay particular attention to this point, and also remove the presence of all solubility/ affinity tags to leave the protein free of any additional amino acids which may alter the stability of the protein; something undesirable when assessing the stability of these proteins with links to SCH 23390 HCl aggregation properties. We propose a simplified purification process of isoelectric precipitation followed by cation exchange chromatography for LEN, and with the addition of size exclusion chromatography for SMA, avoiding the use of lysozyme, which can cause complications in purification and confirm using multiple techniques that the proteins produced here have secondary structure consistent with other.