25 research outputs found
Supplementary guidance: listening to staff: Autumn 2017
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
Macro-to-Micro Structural Proteomics: Native Source Proteins for High-Throughput Crystallization
Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography
The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins
Signaling human mismatch repair by the formation of a hMSH2 -hMSH6 sliding DNA clamp
Germline mutations in several of the human DNA mismatch repair genes, including hMSH2, hMLH1, and hMSH6, are associated with Hereditary Non-Polyposis Colon Cancer. The loss of an intact DNA mismatch repair system and the development of a mutator phenotype is believed to result in carcinogenesis through the accumulation of secondary mutations. As mismatched DNA recognition complexes, the hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers are responsible for initiating a DNA repair event by targeting the repair machinery to the site of the DNA mismatch. The hMSH2-hMSH6 heterodimer recognizes single base and small insertion/deletion DNA mismatches while hMSH2-hMSH3 recognizes small and large insertion/deletion DNA mismatches. This thesis provides a detailed analysis of the mismatched DNA binding and DNA stimulated ATPase activities of the hMSH2-hMSH6 heterodimeric protein. The finding that hMSH2-hMSH6 binds heteroduplex DNA in the ADP-bound form and releases it in the ATP-bound form supports the idea that this protein functions as a molecular switch. Recycling of hMSH2-hMSH6 mismatched DNA binding activity is accomplished by an intrinsic ATPase activity, which hydrolyzes ATP to ADP. In the absence of DNA, hMSH2-hMSH6 will hydrolyze a bound ATP and remain in an ADP bound form capable of binding mismatched DNA. Interaction with mismatched DNA stimulates hMSH2-hMSH6 to exchange ADP for ATP, and causes the ATP-bound protein to release the mismatched base pair by diffusing along the DNA backbone. If a free DNA end is available the ATP-bound hMSH2-hMSH6 will dissociate from the DNA lattice and hydrolyze its bound ATP, hence recycling mismatch-binding activity. Analysis of hMSH2-hMSH6 ATPase stimulation by various types of single base and insertion/deletion DNA mismatch substrates reveals a hierarchy of stimulation showing considerable similarity to previously reported mismatched DNA repair efficiencies in human cells. On the other hand, hMSH2-hMSH6 affinity for various DNA mismatches does not quantitatively agree with reported mismatch DNA repair efficiencies and reveals a strong bias for G/T mismatches. As a whole, this work supports a model for the initiation of bi-directional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to recruitment of the repair machinery
MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes.
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Chapter One 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
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Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3)
Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation
DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks
The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using “error-prone” DNA-repair machinery to generate precise edits
DNA repair outcomes at Cas9-mediated double-strand breaks are non-random and sequence-dependent
The repair of site-specific DNA double-stranded breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 has been extensively used to alter gene function in human cells. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the genomic scars resulting from repair of Cas9-dependent DSBs have not been examined in depth. Repair of DSBs in the absence of donor DNA has been thought to result in random, error-prone repair outcomes; here we show that the patterns of DNA repair following Cas9 cutting are non-random and are reproducible across cell lines and reagent delivery methods. Microhomology contributes reproducibly to a portion of the repair landscape at some sites, but does not fully account for the conservation of repair outcomes. Finally, we find that repair outcomes are determined by the protospacer sequence, the region of DNA targeted via Watson-Crick base pairing by the single-guide RNA (sgRNA) spacer, which opens a path to harness these mutagenic DNA-repair outcomes in a controlled manner to deliver precise editing events