Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinitychromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1)for the removal of C-terminusand N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidinetagofMBP-His6 by Factor Xa and HT 15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-HiS6 to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. Oncolumn tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-1 system was superiorto the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-1 system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-1 (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-1 digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-1 digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-l for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions

Engineering Biofouling Resistant Materials Through the Systematic Adaptation of Surface Morphology

Abstract With increasing numbers of antimicrobial‐resistant (AMR) bacteria strains, it becomes essential that new and effective routes to minimizing bacterial infection rates are produced. Superhydrophobic materials show to be effective in reducing the attachment of bacteria due to their unique wetting properties which can minimize the points at which bacteria can initially adhere. Here, the impact of surface design on the anti‐biofouling capabilities of superhydrophobic pillared arrays prepared via photolithography is investigated. By systematically varying pillar spacing, insight is gained into the complex nature of superhydrophobic fouling as well as allowing for optimization of the antifouling performance. The optimal material within is achieved at a pillar spacing of 87.5 µm, which shows over a 3‐log (and gt; 99.9%) reduction in bacterial attachment

Art Therapy Studio Project

Art Therapy Studio Projec

Activity of a nitric oxide-generating wound treatment system against wound pathogen biofilms

This work was supported by Edixomed Ltd

Antimicrobial Activity Does Not Predict Cytokine Response to Adrenomedullin or Its Shortened Derivatives

The aim of this study was to investigate cytokine release from oral keratinocytes and fibroblasts in response to AM and shortened derivatives previously characterised in terms of their antimicrobial activities. Cells were incubated with AM or its fragments (residues 1-12, 1-21, 13-52, 16-21, 16-52, 22-52, 26-52, and 34-52), and culture supernatants collected after 1, 2, 4, 8, and 24 hours. A time-dependant increase in production of interleukin1-α and interleukin 1-β from keratinocytes in response to all peptides was demonstrated. However, exposure to fragments compared to whole AM resulted in reduced production of these cytokines (60% mean reduction at 24 hours, P<.001). No consistent differences were shown between the cytokine response elicited by antimicrobial and nonantimicrobial fragments. The production of interleukin-6 and interleukin-8 did not change significantly with time or peptide used. Fibroblast cells were relatively unresponsive to all treatments. This study demonstrates that antimicrobial activity does not predict cytokine response to adrenomedullin or its shortened derivatives

The Relationship between both Partial and Complete Denture Wearers and the Presence of Oral Malodour and the Effect of Denture Cleansers on the Oral Microbiota

Oral malodour may be considered a substantial concern for a sizeable percentage of the general population and as such it is important for clinicians to identify the causes of oral malodour, to treat the problem effectively. Aim: The aim of the present study was therefore to review the published literature on the presence and perception of oral malodour (halitosis) in patients wearing both removable partial or complete dentures and the effect of denture cleansers on the oral microbiota. Materials & Methods: A comprehensive electronic search of databases such as PUBMED, Cochrane, Google Scholar, EmBase and Web of Science was performed up to February 2016. Results: 55 potentially relevant reports were identified with six studies included in the review. Of the six included papers, only one study was randomised, and five studies were either non-randomised controlled clinical trials or, quasi randomised trials. The results from these studies would suggest that there was an indirect association between the presence and perception of oral malodour in both RPD and complete denture wearing patients. Conclusions: The strength of evidence was however insufficient to draw any definitive conclusions on a potential correlation of oral malodour in patients with RPDs

Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis

This work was financially supported by the National Natural Science Foundation (No. 81301329, No. 81271957 and No. 81530051), Joint foundation of Liaoning Province Natural Science Foundation and Shenyang National Laboratory for materials science (No. 2015021004), Youth Innovation Promotion Association, CAS (No. 2014168), and The Dunhill Medical Trust (R360/0514)