A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

Facilitators of HIV Medical Care Engagement Among Former Prisoners

Linkage to and retention in medical care is a concern for HIV-positive individuals leaving custody settings in the United States. The minimal existing research points to low rates of entry into care in the months following release and lapsed viral control among releasees who are subsequently reincarcerated. We conducted seven small focus group discussions with 27 HIV-positive individuals who were recently incarceration in a California State prison to understand those factors that facilitated linkage to and retention in HIV care following their release. We used a consensual approach to code and analyze the focus group transcripts. Four main themes emerged from the analysis: 1) interpersonal relationships, 2) professional relationships, 3) coping strategies and resources, and 4) individual attitudes. Improving HIV-related outcomes among individuals after their release from prison requires strengthening supportive relationships, fostering the appropriate attitudes and skills, and ensuring access to resources that stabilize daily living and facilitate the process of accessing care