An Open Library of Human Kinase Domain Constructs for Automated Bacterial Expression.

Kinases play a critical role in cellular signaling and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Therapeutics targeting kinases currently account for roughly 50% of cancer drug discovery efforts. The ability to explore human kinase biochemistry and biophysics in the laboratory is essential to designing selective inhibitors and studying drug resistance. Bacterial expression systems are superior to insect or mammalian cells in terms of simplicity and cost effectiveness but have historically struggled with human kinase expression. Following the discovery that phosphatase coexpression produced high yields of Src and Abl kinase domains in bacteria, we have generated a library of 52 His-tagged human kinase domain constructs that express above 2 µg/mL of culture in an automated bacterial expression system utilizing phosphatase coexpression (YopH for Tyr kinases and lambda for Ser/Thr kinases). Here, we report a structural bioinformatics approach to identifying kinase domain constructs previously expressed in bacteria and likely to express well in our protocol, experiments demonstrating our simple construct selection strategy selects constructs with good expression yields in a test of 84 potential kinase domain boundaries for Abl, and yields from a high-throughput expression screen of 96 human kinase constructs. Using a fluorescence-based thermostability assay and a fluorescent ATP-competitive inhibitor, we show that the highest-expressing kinases are folded and have well-formed ATP binding sites. We also demonstrate that these constructs can enable characterization of clinical mutations by expressing a panel of 48 Src and 46 Abl mutations. The wild-type kinase construct library is available publicly via Addgene.

MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes.

Recombinant expression of large, multiprotein complexes is essential and often rate limiting for determining structural, biophysical, and biochemical properties of DNA repair, replication, transcription, and other key cellular processes. Baculovirus-infected insect cell expression systems are especially well suited for producing large, human proteins recombinantly, and multigene baculovirus systems have facilitated studies of multiprotein complexes. In this chapter, we describe a multigene baculovirus system called MacroBac that uses a Biobricks-type assembly method based on restriction and ligation (Series 11) or ligation-independent cloning (Series 438). MacroBac cloning and assembly is efficient and equally well suited for either single subcloning reactions or high-throughput cloning using 96-well plates and liquid handling robotics. MacroBac vectors are polypromoter with each gene flanked by a strong polyhedrin promoter and an SV40 poly(A) termination signal that minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have successfully assembled as many as 10 genes into a single MacroBac vector. Importantly, we have observed significant increases in expression levels and quality of large, multiprotein complexes using a single, multigene, polypromoter virus rather than coinfection with multiple, single-gene viruses. Given the importance of characterizing functional complexes, we believe that MacroBac provides a critical enabling technology that may change the way that structural, biophysical, and biochemical research is done.

Cytochrome 572 is a conspicuous membrane protein with iron oxidation activity purified directly from a natural acidophilic microbial community.

Recently, there has been intense interest in the role of electron transfer by microbial communities in biogeochemical systems. We examined the process of iron oxidation by microbial biofilms in one of the most extreme environments on earth, where the inhabited water is pH 0.5-1.2 and laden with toxic metals. To approach the mechanism of Fe(II) oxidation as a means of cellular energy acquisition, we isolated proteins from natural samples and found a conspicuous and novel cytochrome, Cyt(572), which is unlike any known cytochrome. Both the character of its covalently bound prosthetic heme group and protein sequence are unusual. Extraction of proteins directly from environmental biofilm samples followed by membrane fractionation, detergent solubilization and gel filtration chromatography resulted in the purification of an abundant yellow-red protein. The purified protein has a cytochrome c-type heme binding motif, CxxCH, but a unique spectral signature at 572 nm, and thus is called Cyt(572). It readily oxidizes Fe(2+) in the physiologically relevant acidic regime, from pH 0.95-3.4. Other physical characteristics are indicative of a membrane-bound multimeric protein. Circular dichroism spectroscopy indicates that the protein is largely beta-stranded, and 2D Blue-Native polyacrylamide gel electrophoresis and chemical crosslinking independently point to a multi-subunit structure for Cyt(572). By analyzing environmental genomic information from biofilms in several distinctly different mine locations, we found multiple genetic variants of Cyt(572). MS proteomics of extracts from these biofilms substantiated the prevalence of these variants in the ecosystem. Due to its abundance, cellular location and Fe(2+) oxidation activity at very low pH, we propose that Cyt(572) provides a critical function for fitness within the ecological niche of these acidophilic microbial communities.