Improved Purification Protocol Research Articles

Crystallographic and biochemical analyses of a far-red allophycocyanin to address the mechanism of the super-red-shift.

Far-red absorbing allophycocyanins (APC), identified in cyanobacteria capable of FRL photoacclimation (FaRLiP) and low-light photoacclimation (LoLiP), absorb far-red light, functioning in energy transfer as light-harvesting proteins. We report an optimized method to obtain high purity far-red absorbing allophycocyanin B, AP-B2, of Chroococcidiopsis thermalis sp. PCC7203 by synthesis in Escherichia coli and an improved purification protocol. The crystal structure of the trimer, (PCB-ApcD5/PCB-ApcB2)3, has been resolved to 2.8Å. The main difference to conventional APCs absorbing in the 650-670nm range is a largely flat chromophore with the co-planarity extending, in particular, from rings BCD to ring A. This effectively extends the conjugation system of PCB and contributes to the super-red-shifted absorption of the α-subunit (λmax = 697nm). On complexation with the β-subunit, it is even further red-shifted (λmax, absorption = 707nm, λmax, emission = 721nm). The relevance of ring A for this shift is supported by mutagenesis data. A variant of the α-subunit, I123M, has been generated that shows an intense FR-band already in the absence of the β-subunit, a possible model is discussed. Two additional mechanisms are known to red-shift the chromophore spectrum: lactam-lactim tautomerism and deprotonation of the chromophore that both mechanisms appear inconsistent with our data, leaving this question unresolved.

Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids.

Many cryogenic electron microscopy (cryo-EM) single particle analyses are constrained by the sample preparation step upon which aggregation, dissociation, and/or preferential orientation of particles can be introduced. Here, we report how we solved these problems in the case of CDC48A, a hexameric AAA ATPase from Arabidopsis thaliana. CDC48A hexamers are well preserved under negative staining conditions but disassemble during grid freezing using the classical blotting method. Vitrification of grids using the blot-free Chameleon method preserved the integrity of particles but resulted in their strong preferential orientation. We then used a strategy where we improved in parallel the purification of CDC48A and the conditions for cryo-EM data acquisition. Indeed, we noted that images taken from thicker ice presented an even distribution of intact particles with random orientations, but resulted in a lower image resolution. Consequently, in our case, distribution, orientation, image resolution, and the integrity of particles were tightly correlated with ice thickness. By combining the more homogeneous and stable CDC48A hexamers resulting from our improved purification protocol with an iterative search across different ice thicknesses, we identified an intermediate thickness that retained sufficiently high-resolution structural information while maintaining a complete distribution of particle orientations. Our approach may provide a simple, fast, and generally applicable strategy to record data of sufficient quality under standard laboratory and microscope settings. This method may be of particular value when time and resources are limited.

Modified secreted alkaline phosphatase as an improved reporter protein for N-glycosylation analysis.

N-glycosylation is a common posttranslational modification of proteins in eukaryotic cells. The modification is often analyzed in cells which are able to produce extracellular, glycosylated proteins. Here we report an improved method of the use of genetically modified, secreted alkaline phosphatase (SEAP) as a reporter glycoprotein which may be used for glycoanalysis. Additional N-glycosylation sites introduced by site-directed mutagenesis significantly increased secretion of the protein. An improved purification protocol of recombinant SEAP from serum or serum-free media is also proposed. The method enables fast and efficient separation of reporter glycoprotein from a relatively small amount of medium (0.5-10 ml) with a high recovery level. As a result, purified SEAP was ready for enzymatic de-glycosylation without buffer exchange, sample volume reductions or other procedures, which are usually time-consuming and may cause partial loss of the reporter glycoprotein.

Short single‐stranded DNA with putative non‐canonical structures comprises a novel class of plasma cell‐free DNA

Cell-free DNA (cfDNA) in human blood is currently being investigated as a minimally invasive, highly informative biomarker. Besides carrying genetic variations, cfDNA can also provide useful epigenetic information through its methylation and fragmentation pattern. Previous cfDNA studies have focused on nucleosome-sized fragments, leaving shorter ones neglected. Hence, we intended to investigate whether shorter cfDNA fragments exist in the blood or not. Using an improved cfDNA purification protocol and a 3ʹ-end labeling method, we found that human plasma contains DNA fragments of approximately 50 nucleotides, present at a molar concentration largely comparable to that of the nucleosome-sized fragments. These short fragments cannot be recovered by using popular cfDNA isolation methods, and they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. We established a library preparation protocol based on a ssDNA ligation technique unique developed by us and applied it to cfDNA isolated with the improved purification protocol. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of these short fragments. Hence, we propose a novel class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

A novel strategy for the preparation of the injectable PET/CT radiopharmaceutical (-)-[11C]-(1R,2S)-meta-hydroxyephedrine ((-)-[11C]HED

Positron emission tomography (PET) had been applied in clinical early diagnosis of various tumors and other diseases. The methylated synthetic conditions of (-)-[11C]-(1R,2S)-meta-hydroxyephedrine ((-)-[11C]HED), considered as one of the most important radiopharmaceuticals for PET, were optimized through single factor and orthogonal design methods. Here, we reported an improved purification protocol. The radiochemical yields of the final product were over 45% (decay-corrected and based on [11C]methyl iodide) (n = 50). The radiochemical purities and chemical purities were over 99% (n = 50) and 97% (n = 50), respectively. The automatic radiosynthesis procedure of (-)-[11C]HED with relatively high radiochemical yield was convenient and reliable.

The Solution Structure and Dynamics of Full-length Human Cerebral Dopamine Neurotrophic Factor and Its Neuroprotective Role against α-Synuclein Oligomers

Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9-107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10-100 and 111-157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1-9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.

Preparation of cysteine-34–nitroxide spin labeled human α1-microglobulin

α1-Microglobulin (α1m) is a protein of yet unresolved function occurring in blood plasma and urine. It consists of a lipocaline type of fold with two cysteine residues forming a disulfide bridge and the third cysteine-34 remaining a free, somewhat reactive thiol. A number of investigations point to an interaction with heme and we have recently reported, that heme binding triggers the formation of a stable α1m trimer upon modification of cysteine-34 with 2-iodoacetamide, i.e., [α1m(heme)2]3 [J.F. Siebel, R.L. Kosinsky, B. Åkerström, M. Knipp, Insertion of heme b into the structure of the Cys34-carbamidomethylated human lipocalin α1-microglobulin—formation of a [(heme)2(α1-microglobulin)]3 complex, ChemBioChem 13 (2012) 879–887]. For further structural and functional investigations, an improved purification protocol for α1m was sought, in particular yielding an untagged amino acid sequence. The method reported herein improves the speed and the yield of the protein production even when an expression plasmid without tag was applied. Furthermore, for the purpose of future structural studies using electron paramagnetic resonance (EPR) techniques, in accordance to the modification with 2-iodoacetamide (α1mAM), the protein was modified with 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-(2-iodoacetamido)-PROXYL) yielding the nitroxide spin labeled α1mN–O. The extinction coefficient of the protein was calibrated using magnetic circular dichroism (MCD) spectroscopy of tryptophan (ε280nm=40,625M−1cm−1). The parallel quantification by absorbance spectroscopy (protein) and cw-EPR spectroscopy (radical spin) determined the degree of spin labeling to 90%. Characterization of the protein by circular dichroism (CD) spectroscopy and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) upon tryptic digestion further demonstrated the similar fold of α1mAM and α1mN–O, but also established the modification of cystein-34 as well as the formation of the cysteine-72–cysteine-169 disulfide bond.

A method for the analysis of theδ18O of inorganic phosphate extracted from soils with HCl

The oxygen isotope composition of phosphate (δ18O‐PO4) has successfully been used to study the biological cycling of phosphorus (P) in seawater and marine sediments. However, only a few studies have used this approach in soils. In order to analyseδ18O‐PO4, phosphate must be extracted from the soil, purified and converted to silver phosphate (Ag3PO4). The published extraction methods, successfully applied to marine waters and sediments, lead to the precipitation of impure Ag3PO4when used with soils or organic‐rich samples. Here we present an improved purification protocol, designed for soils and other organic‐rich samples. After extraction with HCl, phosphate is purified with multiple mineral precipitations that do not require extreme pH adjustments of the solutions. We show that contaminant‐free Ag3PO4can be produced from fertilizers and various soils with different chemical and physical characteristics. Our first isotopic results confirm that differences in P status and availability in soils are expressed in theδ18O‐PO4signal, indicating the potential of this isotopic tracer to understand P dynamics in soil systems.

Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females

The aim of the present study was to address the involvement of Rhipicephalus microplus larval cysteine endopeptidase (RmLCE) in protein digestion in R. microplus larvae and adult females. In this work, an improved purification protocol for native RmLCE was developed. Partial amino acid sequence of the purified enzyme indicates that it is the same enzyme as Boophilus microplus cathepsin-L1 (BmCL1). When vitellin (Vt) degradation by egg and larval enzymes was analyzed, stage-specific differences for RmLCE activity in comparison to vitellin-degrading cysteine endopeptidase (VTDCE) were observed. RmLCE is also able to degrade host hemoglobin (Hb). In agreement, an acidic cysteine endopeptidase activity was detected in larval gut. It was shown that cysteine and aspartic endopeptidases are involved in Vt and Hb digestion in R. microplus larvae and females. Interestingly, we observed that the aspartic endopeptidase Boophilus yolk cathepsin (BYC) is associated with a cysteine endopeptidase activity, in larvae. Synergic hemoglobin digestion by BYC and RmLCE was observed and indicates the presence of an Hb-degrading enzymatic cascade involving these enzymes. Our results suggest that RmLCE/BmCL1 has a continued role in vitellin and hemoglobin digestion during tick development.

Homogeneous recombinant Mycobacterium tuberculosis shikimate dehydrogenase production: An essential step towards target-based drug design

Mycobacterium tuberculosis shikimate dehydrogenase ( MtbSD) catalyzes the forth reaction in the shikimate pathway. Here we describe production of K69A, K69H, K69I, K69Q, D105A, and D105N mutant proteins. Screening of several conditions was performed to optimize MtbSD production yield, and an improved purification protocol to obtain homogeneous MtbSD is presented. The rational design of new antitubercular drugs hinges on the availability of M. tuberculosis proteins. Our results show that optimization of expression, disruption, and purification protocols resulted in a higher yield of functional MtbSD enzyme.

Improved purification protocol for wild-type and mutant human foamy virus proteases

Wild-type and an active site mutant (S25T) human foamy virus (HFV) proteases were expressed in fusion with maltose binding protein in Escherichia coli. The mutant enzyme contained a Ser to Thr mutation in the -Asp-Ser-Gly- active site triplet of the enzyme, which forms the “fireman’s grip” between the two subunits of the homodimeric enzyme. The fusion proteins were purified by affinity chromatography on amylose resin, cleaved with factor Xa, and the processed enzymes were purified by gel filtration under denaturing condition. Refolding after purification resulted in active enzymes with comparable yields. Furthermore, both enzymes showed similar catalytic activities in an oligopeptide substrate representing an HFV Gag cleavage site. However, the S25T mutant showed increased stability in urea unfolding experiment, in a good agreement with the suggested role of the Thr residue of fireman’s grip.

Cloning and purification of the rainbow trout fifth component of complement (C5)

To gain further insight into the evolutionary history of the complement proteins C3, C4, and C5 we have now cloned the fifth component of complement from a rainbow trout ( Oncorhynchus mykiss) liver cDNA library; this is the first report of C5 cloning in a species other than human and mouse. The deduced amino acid sequence of a partial cDNA clone (2.25 kb), representing approximately 44% of the coding sequence, showed 60 and 58% similarity to human and mouse C5, respectively. To validate the molecular information derived from the cloning we developed an improved purification protocol. Mass spectrometric analysis of C5 tryptic digests yielded peptide signals that matched theoretical protein sequence derived from the partial cDNA. Northern blot analysis of RNA from various tissues showed the presence of a single mRNA transcript in trout liver and Southern blot analysis indicated that the gene coding for C5 is present as a single copy in the trout genome. The presence of C5 in trout suggests that C3, C4, and C5 must have diverged before the appearance of teleost fish.

The Lysine-Specific Proteinase from Armillaria mellea Is a Member of a Novel Class of Metalloendopeptidases Located in Basidiomycetes

The fruiting body of the basidiomycete fungus Armillaria mellea produces a lysine-specific proteinase which exhibits both potent fibrinolytic activity and a remarkable resistance to denaturing agents. An improved purification protocol has been developed for this enzyme and the sequence of the 26 N-terminal amino acid residues of the pure protein has been determined by gas-phase sequencing. Searches of the SwissProt database showed that the N-terminal sequence of A. mellea proteinase is highly similar to those of lysine-specific metalloendopeptidases from the basidiomycetes Grifola frondosa and Pleurotus ostreatus. These results support the view that the A. mellea proteinase is a member of a novel class of lysine-specific metalloendopeptidases which may be exclusive to basidiomycete fungi.

Structure of the bifunctional inhibitor of trypsin and alpha-amylase from ragi seeds at 2.9 A resolution.

The crystal structure of a bifunctional inhibitor of alpha-amylase and trypsin from the seeds of ragi (Indian finger millet, Eleusine coracana Gaertneri) has been determined by an X-ray diffraction method. The inhibitor consists of 122 amino acids with five disulfide bridges and belongs to the plant alpha-amylase/trypsin-inhibitor family. This is the first crystal structure determination of a member of this family. The protein, purified from the seeds of ragi, has a molecular mass of 13300 Da with a pI of 10.3. Crystals were grown by a microdialysis method using ammonium sulfate as precipitant. The improved purification protocol and the modified crystallization conditions enabled reproducible growth of the crystals. The cell parameters are a = 41. 2, b = 47.4, c = 55.9 A. The intensity data were collected to 2.9 A resolution, and the crystal structure was determined using the molecular-replacement method. The structure was refined using the X-PLOR and CCP4 program packages to a conventional R factor of 21%. The structure contains four alpha-helices between residues 19-29, 37-51, 56-65 and 90-95, and two short antiparallel beta-strands between residues 67-70 and 73-75.

An Improved Purification Protocol for Heart and Skeletal Muscle Calpastatin Reveals Two Isoforms Resulting from Alternative Splicing

Employment of a new protocol for efficient purification of calpastatin revealed the presence of two forms of calpastatin in porcine heart with apparent molecular weights of 125 and 145 kDa. Purification from bovine muscle resulted in a single species with an apparent molecular weight of 125 kDa. The presence of multiple species of calpastatin in porcine heart does not appear to be an artifact of the purification procedure since Western blotting revealed the presence of two types of calpastatin in ovine and porcine heart, but only one type in bovine heart and bovine, ovine, and porcine muscle. The origin of the two species in porcine heart was examined by RT-PCR and direct sequencing of calpastatin cDNA. This analysis revealed that porcine skeletal muscle exclusively produces transcripts lacking exon 3, while porcine heart produces transcripts that include or lack exon 3, consistent with the presence of two isoforms of the protein. The 125-kDa form of porcine calpastatin therefore appears to be a result of alternative splicing of the calpastatin transcript. The biological significance of the heart specific isoform is not clear; however, its ability to inhibit μ- or m-calpain does not differ considerably. The present purification protocol yielded 4.9 and 1.8 mg calpastatin per kg tissue from porcine heart and bovine skeletal muscle, respectively.

Improved purification protocol of the HSV-1 protease catalytic domain, using immunoaffinity

The catalytic domain of herpes simplex virus protease was expressed in baculovirus-infected cells and purified in milligram quantities by ion-exchange and size-exclusion chromatography. The usefulness of this material was limited by the presence of a contaminating proteolytic activity, which caused time-dependent degradation of the protease. As a result we decided to explore an alternative approach to purification. Specific monoclonal antibodies were produced and evaluated by surface plasmon resonance as ligands for immunoaffinity chromatography. One monoclonal antibody, 6H4, was chosen for coupling to an affinity support, and the resulting column allowed us to obtain a pure and stable enzyme. Immunoaffinity chromatography of herpes simplex virus type 1 protease resulted in successful elimination of the contaminating protease activity. Moreover the immunoaffinity column permitted the isolation of stable and pure enzyme in a one-column procedure.

Purification, characterization, and crystallization of Escherichia coli ribokinase.

Ribokinase phosphorylates ribose to form ribose-5-phosphate in the presence of ATP and magnesium. The phosphorylated sugar can enter the pentose phosphate pathway or be used for the synthesis of nucleotides, histidine, and tryptophan. Ribokinase belongs to the PfkB family of carbohydrate kinases, for which no three-dimensional structure is currently known. We describe an improved purification protocol for Escherichia coli ribokinase and give evidence from light-scattering and gel filtration studies that the protein forms a dimer in solution. Several types of crystals are also described that have been obtained of apo ribokinase, ribokinase in the presence of ATP, and in a ternary complex with an ATP-analogue and ribose. The latter crystals give the best X-ray diffraction. A complete data set has been collected at the synchrotron source in Hamburg, to 2.6 A resolution using a frozen crystal. The crystals belong to space group P6(1)22 or P6(5)22 with cell parameters a = b = 95 A and c = 155 A.

Improved purification protocol of the HSV-1 protease catalytic domain, using immunoaffinity

Improved purification protocol of the HSV-1 protease catalytic domain, using immunoaffinity

Galectin-1 from bovine spleen: biochemical characterization, carbohydrate specificity and tissue-specific isoform profiles.

Selected biochemical properties, including the charge heterodispersity profile and carbohydrate specificity, of bovine galectin-1 were determined in detail. The lectin was purified through an improved purification protocol that yielded 35-40 mg/kg of wet tissue with a specific activity of 1.7-2 x 10(4) mg-1.ml. The galectin is a homodimer of approximately 14.5 kDa subunits with E(280)mg/ml of 0.65 ml.mg-1.cm-1. When stored in the presence of its carbohydrate ligand, the lectin's binding activity remained stable in a non-reducing environment even at room temperature. The optimal pH for binding to the ligand was 6.5-8.0. The overall carbohydrate specificity of the bovine galectin-1 isolated from spleen is similar to that of the galectin isolated from heart and to other mammalian galectins that exhibit "conserved" (Type I) carbohydrate recognition domains (CRDs) [Ahmed, H. and Vasta, G.R. (1994) Glycobiology 4, 545-549], but differs from those from Xenopus laevis and rat intestine domain I. The fluorescence of 4-methylumbelliferyl alpha-D-galactopyranoside was quenched on binding to bovine spleen galectin-1. Scatchard plots of data obtained at 5, 15, and 30 degrees C showed that the galectin has two sugar exothermic binding sites with association constants of 3.4 x 10(5), 1.0 x 10(5), and 0.3 x 10(5), respectively. Chemical modification studies indicated that histidine, tryptophan, carboxylic acid, and arginine, but not lysine or tyrosine, are involved in the binding to the carbohydrate ligand. On isoelectric focusing, the spleen galectin-1 appeared as six isoforms ranging from pI4.56-4.88 with main components at pI 4.63 (34.0%), 4.73 (42.6%), and 4.88 (16.6%). The galectin-1 isolated from heart yielded a quali- and quantitatively different profile with four isoforms ranging from pI 4.53-4.73, those with pIs of 4.56, 4.63, and 4.73 being common to the spleen homolog. Edman degradation of selected peptides purified from the spleen galectin-1 digest revealed amino acid sequences identical to those obtained for the heart galectin-1. This suggests that although point mutations in the subunit primary structure may not be the likely source of isolectins, as observed for X. laevis, tissue-specific co- or post-translational modifications may be the possible cause of the differences in the galectin isoform profile between bovine spleen and heart.

A Fluorometric Assay for Measurement of Mono-ADP-Ribosyltransferase Activity

Using 1,N6-etheno NAD, a fluorescent analog of NAD, we extended an existing assay for NAD glycohydrolase to the measurement of mono-ADP-ribosyltransferase (mADP-RT) activity using agmatine as acceptor for ADP-ribose. The reaction products were analyzed by reversed-phase chromatography. In the presence of agmatine two newly formed fluorescent products were tentatively identified as ADP-ribosylagmatine anomers. Fluorescence intensity increased upon splitting theN-glycoside bondage of 1,N6-etheno NAD. Therefore, 1,N6-etheno AMP could be used for calibration. The nonradioactive assay yielded values nearly identical to those obtained with the [carbonyl-14C]NAD method. It proved to be highly reproducible, rapid, and suitable for an improved purification protocol yielding a 76,000-fold enriched mADP-RT preparation from rabbit skeletal muscle. The identity and high purity of the enzyme were confirmed immunochemically. The assay served to determine the pH optimum of the enzyme (pH 9.0) and itsKMfor 1,N6-etheno NAD (287 μm).