Experimental mapping of soluble protein domains using a hierarchical approach

摘要: Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain in a form suitable for structure determination. Screening methods capable selecting well-expressed, soluble fragments from DNA libraries exist, but require use automation maximize chances picking few good candidates. Here, we describe an insertion dihydrofolate reductase (DHFR) vector select in-frame split-GFP assay technology filter-out constructs that express insoluble fragments. With incorporation IPCR step create high density, focused sublibraries fragments, this cost-effective method can be performed manually with no priori knowledge domain boundaries while permitting single amino acid resolution boundary mapping. We used it on well-characterized p85α subunit phosphoinositide-3-kinase demonstrate robustness efficiency our methodology. then successfully tested onto polyketide synthase PpsC Mycobacterium tuberculosis, potential drug target involved biosynthesis complex lipids cell envelope. X-ray quality crystals acyl-transferase (AT), dehydratase (DH) enoyl-reductase (ER) domains have been obtained.