Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function

A Queueing Theoretic Approach to Decoupling Inventory

This paper investigates the performance of different hybrid push-pull systems with a decoupling inventory at the semi-finished products and reordering thresholds. Raw materials are ‘pushed’ into the semi-finished product inventory and customers ‘pull’ products by placing orders. Furthermore, production of semi-finished products starts when the inventory goes below a certain level, referred to as the threshold value and stops when the inventory attains stock capacity. As performance of the decoupling stock is critical to the overall cost and performance of manufacturing systems, this paper introduces a Markovian model for hybrid push-pull systems. In particular, we focus on a queueing model with two buffers, thereby accounting for both the decoupling stock as well as for possible backlog of orders. By means of numerical examples, we assess the impact of different reordering policies, irregular order arrivals, the set-up time distribution and the order processing time distribution on the performance of hybrid push-pull systems

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR