A custom ÄKTA avant configuration enabling automated parallel protein purification over a range of process scales.

Biologics are making up an increasing proportion of the global drug discovery pipeline. Supporting the expansion of biologics drug discovery requires higher throughput techniques for the expression, purification and characterization of both therapeutic candidates and reagents. Here we describe the programming and development of a novel ÄKTA™ instrument configuration that enables automated parallel and multistep chromatography over a range of scales. The programming strategy is offered as open source and the custom plumbing configuration was developed with off the shelf components available from Cytiva. Combined with high flow resin technology we show how this strategy can reduce the duration of a standard antibody purification process by 4.5X, from 4.5 h down to 1 h per run. An automated loading strategy was also developed to enable true walk away application of up to 24 samples and around the clock processing capability. The techniques used here to accomplish parallel multistep chromatography can be duplicated or modified for specific applications and represent a straightforward and cost-effective means to eliminate protein purification bottlenecks.

Surface interactome in Streptococcus pyogenes.

Very few studies have so far been dedicated to the systematic analysis of protein interactions occurring between surface and/or secreted proteins in bacteria. Such interactions are expected to play pivotal biological roles that deserve investigation. Taking advantage of the availability of a detailed map of surface and secreted proteins in Streptococcus pyogenes (group A Streptococcus (GAS)), we used protein array technology to define the "surface interactome" in this important human pathogen. Eighty-three proteins were spotted on glass slides in high density format, and each of the spotted proteins was probed for its capacity to interact with any of the immobilized proteins. A total of 146 interactions were identified, 25 of which classified as "reciprocal," namely, interactions that occur irrespective of which of the two partners was immobilized on the chip or in solution. Several of these interactions were validated by surface plasmon resonance and supported by confocal microscopy analysis of whole bacterial cells. By this approach, a number of interesting interactions have been discovered, including those occurring between OppA, DppA, PrsA, and TlpA, proteins known to be involved in protein folding and transport. These proteins, all localizing at the septum, might be part, together with HtrA, of the recently described ExPortal complex of GAS. Furthermore, SpeI was found to strongly interact with the metal transporters AdcA and Lmb. Because SpeI strictly requires zinc to exert its function, this finding provides evidence on how this superantigen, a major player in GAS pathogenesis, can acquire the metal in the host environment, where it is largely sequestered by carrier proteins. We believe that the approach proposed herein can lead to a deeper knowledge of the mechanisms underlying bacterial invasion, colonization, and pathogenesis.

The mechanism of membrane-associated steps in tail-anchored protein insertion.

Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle.