Rapid purification of tRNA(Lys) from rat liver

Fast protein liquid chromatography (FPLC) system using Mono Q (HR 5/5) anion-exchange column chromatography followed by highly cross-linked urea-polyacrylamide gel electrophoresis (urea-PAGE) was used for the purification of lysine-specific tRNA (tRNA(Lys)) from rat liver. Crude tRNA from rat liver was fractionated with a linear gradient of NaCl (0.3-0.8 M) in triethanolamine-HCl buffer, pH 4.5, and the activity of tRNA(Lys) was found to elute between 0.51 and 0.57 M NaCl. Using this concentration range of NaCl, tRNA(Lys) was refractionated on the same column with a shallow gradient, where a single peak of tRNA(Lys) activity was obtained. tRNA(Lys)-rich fractions recovered from the second run were electrophoretically separated on 16% polyacrylamide-7 M urea gel into one major band and three minor bands. The major band showed a specific activity of 997 pmols/A260 U for tRNALys with a 43-fold purification and approximately 17% recovery. The minor bands displayed negligible or no activity for lysine. tRNA(Lys) obtained by this method was found to be homogeneous by competitive aminoacylation. The advantages of FPLC followed by urea-PAGE in the purification of an amino acid-specific tRNA over conventional column chromatography are discussed.

Thermolabile ribonucleases from antarctic psychrotrophic bacteria: detection of the enzyme in various bacteria and purification from Pseudomonas fluorescens

Thirteen terrestrial psychrotrophic bacteria from Antarctica were screened for the presence of a thermolabile ribonuclease (RNAase-HL). The enzyme was detected in three isolates of Pseudomonas fluorescens and one isolate of Pseudomonas syringae. It was purified from one P. Fluorescens isolate and the molecular mass of the enzyme as determined by SDS-PAGE was 16 kDa. RNAase-HL exhibited optimum activity around 40 degrees C at pH 7.4. It could hydrolyse Escherichia coli RNA and the synthetic substrates poly(A), poly(C), poly(U) and poly(A-U). Unlike the crude RNAase from mesophilic P. Fluorescens and pure bovine pancreatic RNAase A which were active even at 65 degrees C, RNAase-HL was totally and irreversibly inactivated at 65 degrees C.

CDNA Cloning, Overexpression in Escherichia coli, Purification and Characterization of Sheep Liver Cytosolic Serine Hydroxymethyltransferase

A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/l bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme. The K-m values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5'-phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5'-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme.

Cloning, expression, purification, crystallization and preliminary X-ray studies of the mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis

The mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis has been cloned, expressed, purified and crystallized and the crystals have been characterized using X-ray diffraction. The Matthews coefficient suggests the possibility of two lectin domains in the triclinic cell. The amino-acid sequence of the domain indicates structural similarity to well characterized beta-prism II fold lectins.

Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization

Chicken egg yolk biotin-binding protein-I (BBP-I) has been purified to homogeneity along with the tetrameric BBP-II by a common protocol. The purification includes delipidation of egg yolk by butanol extraction, DEAE-Sephacel chromatography, treatment with guanidinium chloride and biotin-aminohexyl-Sepharose affinity chromatography. The identity of purified BBP-I was ascertained by its physicochemical properties as well as by its immunological cross-reactivity and precursor-product relationship with BBP-II.

Purification and characterization of DNA-dependent RNA polymerase II from Candida utilis

DNA-dependent RNA polymerase II from Candida utilis has been purified to near homogeneity. The purified enzyme resolved into three subforms, viz. IIO, IIA and IIB. On SDS-PAGE the enzyme showed ten polypeptides with molecular weights in the range of 205 kDa to 14 kDa. By two dimensional electrophoresis (IEF followed by SDS-PAGE) the presence of basic and acidic polypeptides has been demonstrated. The enzyme showed Km values of 5, 5.6 and 8 mu M for GTP, CTP and ATP, respectively, and the activity was inhibited by low levels of oc-amanitin and antibodies raised against bovine RNA polymerase II. By Western blot analysis the enzyme was found to cross-react with antibodies to bovine RNA polymerase II. RNA polymerase II from G. utilis is a phosphoprotein, the subunits RPB1 and RPB10 were found to be phosphorylated. Analysis of carboxy-terminal domain indicated that it was functionally redundant at least in case of nonspecific transcription, implicating its role in other nuclear processes, such as promoter specific initiation or transcription activation or RNA processing.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea)

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C-4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-C-14]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparision of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

Vitellins and vitellogenins of Dysdercus koenigii (Heteroptera : Pyrrhocoridae) - identification, purification and temporal pattern

Two vitellins, VtA and VtB, were purified from the eggs of Dysdercus koenigii by gel filtration and ion exchange chromatography. VtA and VtB have molecular weights of 290 and 260 kDa, respectively. Both Vts are glycolipoproteinaceous in nature. VtA is composed of three polypeptides of M-r 116, 92 and 62 kDa while VtB contained an additional subunit of M-r 40 kDa. All subunits except the 116-kDa subunit are glycolipopolypeptides. Polyclonal antibody raised against VtA (anti-VtA antibody) cross-reacted with VtB and also with vitellogenic haemolymph and ovaries and pre-vitellogenic fat bodies, but not with haemolymph from either adult male, fifth instar female, or pre-vitellogenic females demonstrating sex and stage specificity of the Vts. Immunoblots in the presence of anti-VtA revealed two proteins (of 290 and 260 kDa) in both vitellogenic haemolymph and pre-vitellogenic fat bodies that are recognised as D. koenigii Vgs. In newly emerged females, Vgs appeared on day 1 in fat bodies and on day 3 in haemolymph and ovaries. Vg concentration was maximum on day 2 in fat body, day 4 in haemolymph and day 7 in ovary. Although the biochemical and temporal characteristics of these proteins show similarity to some hemipterans, they are strikingly dissimilar with those of a very closely related species. (C) 1999 Elsevier Science Inc. All rights reserved.

Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to 2.5 angstrom resolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme.

Affinity-Chromatographic Procedure For The Purification Of The Enzyme From Mung-Bean (Phaseolus, Aureus) Seeds And Conformational-Changes On Its Interaction With Ortho-Phosphate

Flavokinase was purified, for the first time from a plant source [mung bean (Phaseolus aureus)] by affinity chromatography in the presence of orthophosphate and by using C-8 ATP-agarose (ATP linked through the C-8 position to beaded agarose), Cibacron Blue and riboflavin--Sepharoses. An altered substrates-saturation pattern was observed in the presence of K2HPO4. The conformational changes of the enzyme in the presence of K2HPO4 were monitored by fluorescence spectroscopy. These results highlight the regulatory nature of this enzyme.

Identification and purification of tRNAs containing N6-(delta 2-isopentenyl) adenosine using antibodies specific for N6-(delta-isopentenyl) adenosine

Antibodies specific for the modified nucleoside N6-(delta 2-isopentenyl) adenosine (i6A) were employed to identify the tRNAs containing i6A from an unfractionated tRNA mixture by a nitrocellulose filter binding assay. When radioactive aminoacyl-tRNAs were incubated with i6A-specific antibodies and filtered through nitrocellulose membrane filters, the tRNAs possessing i6A (tRNAtyr and tRNAser) remained on the filters. tRNAarg and tRNAlys which do not contain i6A showed no binding. This finding will be useful as a very simple and rapid assay of such RNAs under a variety of conditions. Purification of i6A containing tRNAs from an unfractionated tRNA mixture was achieved by affinity chromatography of the tRNAs on an i6A antibody-Sepharose column. Nonspecific binding of tRNAs to the column was avoided by the use of purified antibodies.

Smart polymer mediated purification and recovery of active proteins from inclusion bodies

Obtaining correctly folded proteins from inclusion bodies of recombinant proteins expressed in bacterial hosts requires solubilization with denaturants and a refolding step. Aggregation competes with the second step. Refolding of eight different proteins was carried out by precipitation with smart polymers. These proteins have different molecular weights, different number of disulfide bridges and some of these are known to be highly prone to aggregation. A high throughput refolding screen based upon fluorescence emission maximum around 340 nm (for correctly folded proteins) was developed to identify the suitable smart polymer. The proteins could be dissociated and recovered after the refolding step. The refolding could be scaled up and high refolding yields in the range of 8 mg L-1 (for CD4D12, the first two domains of human CD4) to 58 mg L-1 (for malETrx, thioredoxin fused with signal peptide of maltose binding protein) were obtained. Dynamic light scattering (DLS) showed that polymer if chosen correctly acted as a pseuclochaperonin and bound to the proteins. It also showed that the time for maximum binding was about 50 min which coincided with the time required for incubation (with the polymer) before precipitation for maximum recovery of folded proteins. The refolded proteins were characterized by fluorescence emission spectra, circular dichroism (CD) spectroscopy, melting temperature (T-m), and surface hydrophobicity measurement by ANS (8-anilinol-naphthalene sulfonic acid) fluorescence. Biological activity assay for thioredoxin and fluorescence based assay in case of maltose binding protein (MBP) were also carried out to confirm correct refolding. (C) 2012 Elsevier B.V. All rights reserved.