This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His6-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single beta-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory informationmanagement system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

beta-Subunits of voltage-dependent Ca(2+) channels regulate both their expression and biophysical properties. We have injected a range of concentrations of beta3-cDNA into Xenopus oocytes, with a fixed concentration of alpha1B (Ca(V)2.2) cDNA, and have quantified the corresponding linear increase of beta3 protein. The concentration dependence of a number of beta3-dependent processes has been studied. First, the dependence of the a1B maximum conductance on beta3-protein occurs with a midpoint around the endogenous concentration of beta3 (approximately 17 nM). This may represent the interaction of the beta-subunit, responsible for trafficking, with the I-II linker of the nascent channel. Second, the effect of beta3-subunits on the voltage dependence of steady-state inactivation provides evidence for two channel populations, interpreted as representing alpha1B without or with a beta3-subunit, bound with a lower affinity of 120 nM. Third, the effect of beta3 on the facilitation rate of G-protein-modulated alpha1B currents during a depolarizing prepulse to +100 mV provides evidence for the same two populations, with the rapid facilitation rate being attributed to Gbetagamma dissociation from the beta-subunit-bound alpha1B channels. The data are discussed in terms of two hypotheses, either binding of two beta-subunits to the alpha1B channel or a state-dependent alteration in affinity of the channel for the beta-subunit.

The plasma membrane expression of the rat brain calcium channel subunits alpha1A, alpha2-delta and the beta subunits beta1b, beta2a, beta3b and beta4 was examined by transient expression in COS-7 cells. Neither alpha1A nor alpha2-delta localized to the plasma membrane, either alone or when coexpressed. However, coexpression of alpha1A or alpha2-delta/alpha1A with any of the beta subunits caused alpha1A and alpha2 to be targetted to the plasma membrane. The alpha1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native alpha1A channels in dorsal root ganglion neurons and for alpha1A expressed with a beta subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of alpha1A. Immunostaining for alpha1A was obtained in depolarized non-permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a beta subunit. In contrast, beta1b and beta2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target alpha1A to the membrane since beta3 and beta4 alone showed no differential localization, but did direct the translocation of alpha1A to the plasma membrane, suggesting a chaperone role for the beta subunits.

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single beta-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).

MICALs are large (Ͼ1,000 aa), multidomain, cytosolic proteins expressed in specific neuronal and nonneuronal (thymus, lung, spleen, and testis) tissues both during development and in adulthood (4).From sequence analysis, it has been shown that MICALs contain two protein-protein interaction domains implicated in signal transduction and cytoskeletal organization, a calponin homology (CH) domain (7) and a LIM domain (8), plus a proline-rich region for Src homology 3 (SH3) domain recognition that mediates interaction with CasL, a multidomain docking protein localized at focal adhesions and stress fibers (4). Human MICAL-1 associates with the small GTPase Rab1 (6, 9) and with vimentin (4), a major component of intermediate filaments. In addition to the SH3 domainbinding motif, the C-terminal region (of Ϸ250 residues) contains coiled-coil motifs and binds the cytosolic domain of class A plexins (5). Thus, the MICALs are protein-binding scaffolds, but, uniquely, they combine this property with a highly conserved N-terminal region of some 500 residues, characterized by sequence analyses and functional studies as a putative flavoprotein monooxygenase (MO) required for semaphorin-plexin-mediated axon guidance (5).Flavoenzymes bind the cofactor FAD as an integral part of their structure. Despite Ͻ20% sequence identity between disparate members of this family, they share a similar fold and essentially identical FAD-binding sites (10). In contrast, the catalytic reactions carried out by the flavoenzymes are varied, and their active-site architectures differ accordingly. The structure of p-hydroxybenzoate hydroxylase (PHBH) provides the paradigm for the flavoprotein MO (hydroxylase) subset of flavoenzymes (11). Flavoprotein MOs act on a broad range of small molecules (e.g., phydroxybenzoate, steroids, and amino acids). The substrate(s), mode of action, and, indeed, function of the putative MO region in the MICALs are unknown.Our structural and biophysical analyses on the N-terminal portion of murine MICAL-1 confirm that this region has the architecture and characteristics of a flavoenzyme of the MO family, demonstrate the enzymatic activity to be NADPH-dependent, and reveal a mechanism for controlled substrate access to the active site, which is strongly indicative of large (potentially protein) substrates. MethodsProtein Expression and Purification. The mMICAL 489 expression construct (amino acids 1-489 of the mouse MICAL-1 gene plus C-terminal His-tag) was generated by ligation-independent cloning (Gateway Technology, Invitrogen), overexpressed in Escherichia coli (DE3)pLysS (Novagen), and purified with Ni affinity and size-exclusion chromatography; all stages used the high-throughput pipeline of the Oxford Protein Production Facility (see Supporting Text, which is published as supporting information on the PNAS

Structure prediction methods have been used to establish a domain structure for the voltage-dependent calcium channel L L subunit, L L1b. One domain was identified from homology searches as an SH3 domain, whilst another was shown, using threading algorithms, to be similar to yeast guanylate kinase. This domain structure suggested relatedness to the membrane-associated guanylate kinase protein family, and that the N-terminal domain of the L L subunit might be similar to a PDZ domain. Three-dimensional model structures have been constructed for these three domains. The extents of the domains are consistent with functional properties and mutational assays of the subunit, and provide a basis for understanding its modulatory function.z 1999 Federation of European Biochemical Societies.