Chemically cross-linked hydrogels from repetitive protein arrays.

Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype. Our hydrogel design was made possible by the incorporation of the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein that spontaneously formed covalent crosslinks upon mixing. Changing the ratios of the protein building blocks (ST:SC), allowed the viscoelastic properties and gelation speeds of the hydrogels to be altered and controlled. The physical properties of the hydrogels could readily be altered further to suit different environments by tuning the key features in the repetitive protein sequence. The resulting hydrogels were designed with a view to allow cell attachment and encapsulation of liver derived cells. Biocompatibility of the hydrogels was assayed using a HepG2 cell line constitutively expressing GFP. The cells remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. Our results demonstrate how this genetically encoded approach using repetitive proteins could be applied to bridge engineering biology with nanotechnology creating a level of biomaterial customisation previously inaccessible.

The C-terminal helix of BubR1 is essential for CENP-E-dependent chromosome alignment.

During cell division, misaligned chromosomes are captured and aligned by motors before their segregation. The CENP-E motor is recruited to polar unattached kinetochores to facilitate chromosome alignment. The spindle checkpoint protein BubR1 (also known as BUB1B) has been reported as a CENP-E interacting partner, but the extent to which BubR1 contributes to CENP-E localization at kinetochores has remained controversial. Here we define the molecular determinants that specify the interaction between BubR1 and CENP-E. The basic C-terminal helix of BubR1 is necessary but not sufficient for CENP-E interaction, and a minimal key acidic patch on the kinetochore-targeting domain of CENP-E is also essential. We then demonstrate that BubR1 is required for the recruitment of CENP-E to kinetochores to facilitate chromosome alignment. This BubR1-CENP-E axis is critical for alignment of chromosomes that have failed to congress through other pathways and recapitulates the major known function of CENP-E. Overall, our studies define the molecular basis and the function for CENP-E recruitment to BubR1 at kinetochores during mammalian mitosis.This article has an associated First Person interview with the first author of the paper.

An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor.

We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that, within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors, while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer-monomer dissociation Kd is estimated to be ∼0.9 µM with a slow dissociation rate (t1/2 ∼ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such 'allostatic' regulation may be important in metabolic reprogramming and influencing cell fate.

Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation.

We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation, and that these modifications regulate enzyme activity by preventing the formation of the active tetrameric form. The biotin-switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358, and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80, 100, and 140% activity, respectively, compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast, the Cys326Ser mutant exists predominantly in monomeric form. A biotin-switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK, confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer-tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.

Molecular dynamics simulations of site point mutations in the TPR domain of cyclophilin 40 identify conformational states with distinct dynamic and enzymatic properties.

Cyclophilin 40 (Cyp40) is a member of the immunophilin family that acts as a peptidyl-prolyl-isomerase enzyme and binds to the heat shock protein 90 (Hsp90). Its structure comprises an N-terminal cyclophilin domain and a C-terminal tetratricopeptide (TPR) domain. Cyp40 is overexpressed in prostate cancer and certain T-cell lymphomas. The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308, referred to as the carboxylate clamp, and is essential for Cyp40-Hsp90 binding. In this study, the effect of two mutations, K227A and K308A, and their combinative mutant was investigated by performing a total of 5.76 µs of all-atom molecular dynamics (MD) simulations in explicit solvent. All simulations, except the K308A mutant, were found to adopt two distinct (extended or compact) conformers defined by different cyclophilin-TPR interdomain distances. The K308A mutant was only observed in the extended form which is observed in the Cyp40 X-ray structure. The wild-type, K227A, and combined mutant also showed bimodal distributions. The experimental melting temperature, Tm, values of the mutants correlate with the degree of compactness with the K308A extended mutant having a marginally lower melting temperature. Another novel measure of compactness determined from the MD data, the "coordination shell volume," also shows a direct correlation with Tm. In addition, the MD simulations show an allosteric effect with the mutations in the remote TPR domain having a pronounced effect on the molecular motions of the enzymatic cyclophilin domain which helps rationalise the experimentally observed increase in enzyme activity measured for all three mutations.

Novel Entropically Driven Conformation-specific Interactions with Tomm34 Protein Modulate Hsp70 Protein Folding and ATPase Activities.

Co-chaperones containing tetratricopeptide repeat (TPR) domains enable cooperation between Hsp70 and Hsp90 to maintain cellular proteostasis. Although the details of the molecular interactions between some TPR domains and heat shock proteins are known, we describe a novel mechanism by which Tomm34 interacts with and coordinates Hsp70 activities. In contrast to the previously defined Hsp70/Hsp90-organizing protein (Hop), Tomm34 interaction is dependent on the Hsp70 chaperone cycle. Tomm34 binds Hsp70 in a complex process; anchorage of the Hsp70 C terminus by the TPR1 domain is accompanied by additional contacts formed exclusively in the ATP-bound state of Hsp70 resulting in a high affinity entropically driven interaction. Tomm34 induces structural changes in determinants within the Hsp70-lid subdomain and modulates Hsp70/Hsp40-mediated refolding and Hsp40-stimulated Hsp70 ATPase activity. Because Tomm34 recruits Hsp90 through its TPR2 domain, we propose a model in which Tomm34 enables Hsp70/Hsp90 scaffolding and influences the Hsp70 chaperone cycle, providing an additional role for co-chaperones that contain multiple TPR domains in regulating protein homeostasis.

Biophysical Characterization and Activity of Lymphostatin, a Multifunctional Virulence Factor of Attaching and Effacing Escherichia coli.

Attaching and effacing Escherichia coli cause diarrhea and typically produce lymphostatin (LifA), an inhibitor of mitogen-activated proliferation of lymphocytes and pro-inflammatory cytokine synthesis. A near-identical factor (Efa1) has been reported to mediate adherence of E. coli to epithelial cells. An amino-terminal region of LifA shares homology with the catalytic domain of the large clostridial toxins, which are retaining glycosyltransferases with a DXD motif involved in binding of a metal ion. Understanding the mode(s) of action of lymphostatin has been constrained by difficulties obtaining a stably transformed plasmid expression clone. We constructed a tightly inducible clone of enteropathogenic E. coli O127:H6 lifA for affinity purification of lymphostatin. The purified protein inhibited mitogen-activated proliferation of bovine T lymphocytes in the femtomolar range. It is a monomer in solution and the molecular envelope was determined using both transmission electron microscopy and small-angle x-ray scattering. Domain architecture was further studied by limited proteolysis. The largest proteolytic fragment containing the putative glycosyltransferase domain was tested in isolation for activity against T cells, and was not sufficient for activity. Tryptophan fluorescence studies indicated thatlymphostatin binds uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) but not UDP-glucose (UDP-Glc). Substitution of the predicted DXD glycosyltransferase motif with alanine residues abolished UDP-GlcNAc binding and lymphostatin activity, although other biophysical properties were unchanged. The data indicate that lymphostatin has UDP-sugar binding potential that is critical for activity, and is a major leap toward identifying the nature and consequences of modifications of host cell factors.

Inhibition of Antigen-Specific and Nonspecific Stimulation of Bovine T and B Cells by Lymphostatin from Attaching and Effacing Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are enteric bacterial pathogens of worldwide importance. Most EPEC and non-O157 EHEC strains express lymphostatin (also known as LifA), a chromosomally encoded 365-kDa protein. We previously demonstrated that lymphostatin is a putative glycosyltransferase that is important in intestinal colonization of cattle by EHEC serogroup O5, O111, and O26 strains. However, the nature and consequences of the interaction between lymphostatin and immune cells from the bovine host are ill defined. Using purified recombinant protein, we demonstrated that lymphostatin inhibits mitogen-activated proliferation of bovine T cells and, to a lesser extent, proliferation of cytokine-stimulated B cells, but not NK cells. It broadly affected the T cell compartment, inhibiting all cell subsets (CD4, CD8, WC-1, and γδ T cell receptor [γδ-TCR]) and cytokines examined (interleukin 2 [IL-2], IL-4, IL-10, IL-17A, and gamma interferon [IFN-γ]) and rendered T cells refractory to mitogen for a least 18 h after transient exposure. Lymphostatin was also able to inhibit proliferation of T cells stimulated by IL-2 and by antigen presentation using a Theileria-transformed cell line and autologous T cells from Theileria-infected cattle. We conclude that lymphostatin is likely to act early in T cell activation, as stimulation of T cells with concanavalin A, but not phorbol 12-myristate 13-acetate combined with ionomycin, was inhibited. Finally, a homologue of lymphostatin from E. coli O157:H7 (ToxB; L7095) was also found to possess comparable inhibitory activity against T cells, indicating a potentially conserved strategy for interference in adaptive responses by attaching and effacing E. coli.

Protein-Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP).

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control.

A t(1;11) translocation linked to schizophrenia and affective disorders gives rise to aberrant chimeric DISC1 transcripts that encode structurally altered, deleterious mitochondrial proteins.

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a risk factor for psychiatric illness through its disruption by a balanced chromosomal translocation, t(1;11)(q42.1;q14.3), that co-segregates with schizophrenia, bipolar disorder and depression. We previously reported that the translocation reduces DISC1 expression, consistent with a haploinsufficiency disease model. Here we report that, in lymphoblastoid cell lines, the translocation additionally results in the production of abnormal transcripts due to the fusion of DISC1 with a disrupted gene on chromosome 11 (DISC1FP1/Boymaw). These chimeric transcripts encode abnormal proteins, designated CP1, CP60 and CP69, consisting of DISC1 amino acids 1-597 plus 1, 60 or 69 amino acids, respectively. The novel 69 amino acids in CP69 induce increased α-helical content and formation of large stable protein assemblies. The same is predicted for CP60. Both CP60 and CP69 exhibit profoundly altered functional properties within cell lines and neurons. Both are predominantly targeted to mitochondria, where they induce clustering and loss of membrane potential, indicative of severe mitochondrial dysfunction. There is currently no access to neural material from translocation carriers to confirm these findings, but there is no reason to suppose that these chimeric transcripts will not also be expressed in the brain. There is thus potential for the production of abnormal chimeric proteins in the brains of translocation carriers, although at substantially lower levels than for native DISC1. The mechanism by which inheritance of the translocation increases risk of psychiatric illness may therefore involve both DISC1 haploinsufficiency and mitochondrial deficiency due to the effects of abnormal chimeric protein expression. GenBank accession numbers: DISC1FP1 (EU302123), Boymaw (GU134617), der 11 chimeric transcript DISC1FP1 exon 2 to DISC1 exon 9 (JQ650115), der 1 chimeric transcript DISC1 exon 4 to DISC1FP1 exon 4 (JQ650116), der 1 chimeric transcript DISC1 exon 6 to DISC1FP1 exon 3a (JQ650117).

Trypanosomatid phosphoglycerate mutases have multiple conformational and oligomeric states.

Three structurally distinct forms of phosphoglycerate mutase from the trypanosomatid parasite Leishmania mexicana were isolated by standard procedures of bacterial expression and purification. Analytical size-exclusion chromatography coupled to a multi-angle scattering detector detected two monomeric forms of differing hydrodynamic radii, as well as a dimeric form. Structural comparisons of holoenzyme and apoenzyme trypanosomatid cofactor-independent phosphoglycerate mutase (iPGAM) X-ray crystal structures show a large conformational change between the open (apoenzyme) and closed (holoenzyme) forms accounting for the different monomer hydrodynamic radii. Until now iPGAM from trypanosomatids was considered to be only monomeric, but results presented here show the appearance of a dimeric form. Taken together, these observations are important for the choice of screening strategies to identify inhibitors of iPGAM for parasite chemotherapy and highlight the need to select the most biologically or functionally relevant form of the purified enzyme.

The mitosis and neurodevelopment proteins NDE1 and NDEL1 form dimers, tetramers, and polymers with a folded back structure in solution.

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8-167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.

Triazole biotin: a tight-binding biotinidase-resistant conjugate.

The natural amide bond found in all biotinylated proteins has been replaced with a triazole through CuAAC reaction of an alkynyl biotin derivative. The resultant triazole-linked adducts are shown to be highly resistant to the ubiquitous hydrolytic enzyme biotinidase and to bind avidin with dissociation constants in the low pM range. Application of this strategy to the production of a series of biotinidase-resistant biotin-Gd-DOTA contrast agents is demonstrated.

A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase.

PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.

Activity of Lymphostatin, A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli, is Dependent on a Cysteine Protease Motif.

Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wild-type LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine, which inhibit endosome acidification. The cleavage product was not observed in cells treated with the C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin and its activity against T cells.

Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice.

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.

Estimating the affinity of protein-ligand complex from changes to the charge-state distribution of a protein in electrospray ionization mass spectrometry.

The detection of low affinity interactions between proteins and ligands by biophysical methods is challenging. It is often necessary to use competition methods that are time consuming and require well characterized known binders. A mass spectrometry approach is presented for identifying low affinity protein-ligand binding which does not require direct detection of the parent protein-ligand complex but depends on characteristic changes observed in the protein mass spectrum. We observe that on titration of ligand there are characteristic 'charge-state shifts' which manifest as changes in the relative intensities of protein peaks that correlate with the degree of protein-ligand complex formation. We suggest that use of this phenomenon will be particularly suitable for the identification of low affinity complexes where the intensity of any complex ion would be close to noise.

A computationally designed binding mode flip leads to a novel class of potent tri-vector cyclophilin inhibitors.

Cyclophilins (Cyps) are a major family of drug targets that are challenging to prosecute with small molecules because the shallow nature and high degree of conservation of the active site across human isoforms offers limited opportunities for potent and selective inhibition. Herein a computational approach based on molecular dynamics simulations and free energy calculations was combined with biophysical assays and X-ray crystallography to explore a flip in the binding mode of a reported urea-based Cyp inhibitor. This approach enabled access to a distal pocket that is poorly conserved among key Cyp isoforms, and led to the discovery of a new family of sub-micromolar cell-active inhibitors that offer unprecedented opportunities for the development of next-generation drug therapies based on Cyp inhibition. The computational approach is applicable to a broad range of organic functional groups and could prove widely enabling in molecular design.

Thermo-kinetic analysis space expansion for cyclophilin-ligand interactions - identification of a new nonpeptide inhibitor using Biacore™ T200.

We have established a refined methodology for generating surface plasmon resonance sensor surfaces of recombinant his-tagged human cyclophilin-A. Our orientation-specific stabilisation approach captures his-tagged protein under 'physiological conditions' (150 mm NaCl, pH 7.5) and covalently stabilises it on Ni2+-nitrilotriacetic acid surfaces, very briefly activated for primary amine-coupling reactions, producing very stable and active surfaces (≥ 95% specific activity) of cyclophilin-A. Variation in protein concentration with the same contact time allows straightforward generation of variable density surfaces, with essentially no loss of activity, making the protocol easily adaptable for studying numerous interactions; from very small fragments, ~ 100 Da, to large protein ligands. This new method results in an increased stability and activity of the immobilised protein and allowed us to expand the thermo-kinetic analysis space, and to determine accurate and robust thermodynamic parameters for the cyclophilin-A-cyclosporin-A interaction. Furthermore, the increased sensitivity of the surface allowed identification of a new nonpeptide inhibitor of cyclophilin-A, from a screen of a fragment library. This fragment, 2,3-diaminopyridine, bound specifically with a mean affinity of 248 ± 60 µm. The X-ray structure of this 109-Da fragment bound in the active site of cyclophilin-A was solved to a resolution of 1.25 Å (PDB: 5LUD), providing new insight into the molecular details for a potential new series of nonpeptide cyclophilin-A inhibitors.

A Streamlined, Automated Protocol for the Production of Milligram Quantities of Untagged Recombinant Rat Lactate Dehydrogenase A Using ÄKTAxpressTM.

We developed an efficient, automated 2-step purification protocol for the production of milligram quantities of untagged recombinant rat lactate dehydrogenase A (rLDHA) from E. coli, using the ÄKTAxpress™ chromatography system. Cation exchange followed by size exclusion results in average final purity in excess of 93% and yields ~ 14 milligrams per 50 ml of original cell culture in EnPresso B media, in under 8 hrs, including all primary sample processing and column equilibration steps. The protein is highly active and coherent biophysically and a viable alternative to the more problematic human homolog for structural and ligand-binding studies; an apo structure of untagged rLDHA was solved to a resolution 2.29 Å (PDB ID 5ES3). Our automated methodology uses generic commercially available pre-packed columns and simple buffers, and represents a robust standard method for the production of milligram amounts of untagged rLDHA, facilitating a novel fragment screening approach for new inhibitors.