Alzheimer's disease (AD) is the most widespread neurodegenerative disease, strongly linked to amyloid depositions in the brain consisting of amyloid β (Aβ) peptides. The likelihood of developing late- Show more
Alzheimer's disease (AD) is the most widespread neurodegenerative disease, strongly linked to amyloid depositions in the brain consisting of amyloid β (Aβ) peptides. The likelihood of developing late-onset Alzheimer's disease (LOAD) is influenced by the specific isoforms of apolipoprotein E (ApoE), with ApoE4 being the strongest known genetic risk factor for LOAD. Strong evidence suggests that ApoE impacts AD by modulating Aβ aggregation and clearance, although the precise molecular mechanisms remain incompletely understood. Microscale thermophoresis (MST) is a powerful technique for characterizing molecular interactions in solution, which has been used to determine various binding constants, although not the binding of ApoE to Aβ peptides. MST results show that ApoE isoforms bind Aβ1-40 and Aβ1-42 with low micromolar affinity. For Aβ1-42, ApoE3 shows the strongest binding ( Show less
Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binde Show more
Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the "affinity maturation" step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. Show less
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to Show more
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators. Show less