Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function

RNase P is an essential enzyme that processes 5'' end leader sequence of pre-tRNA to generate mature tRNA. The bacterial RNase Ps contain a RNA subunit and one protein subunit, where the RNA subunit contains the catalytic activity. The protein subunit which lacks any catalytic activity, relaxes the ionic requirements for holoenzyme reaction and is indispensable for pre-tRNA cleavage in vivo. In the current study, we reconstituted the M. tuberculosis RNase P holoenzyme in vitro. We prepared the RNase P protein through two different strategies that differ in the conditions under which the recombinant M. tuberculosis protein, expressed in E. coli was purified. The mycobacterial RNase P protein which was purified under native conditions subsequent to isolation from inclusion bodies and in vitro renaturation, was capable of cleaving pre-tRNA specifically without the requirement of RNase P RNA. However, the preparation that was purified under denaturing conditions and refolded subsequently lacked any inherent pre-tRNA processing activity and cleaved the substrate only as a component of the holoenzyme with the RNA subunit. We found that the two RNase P protein preparations attained alternative conformations and differed with respect to their stability as well.     

Matrix-Assisted Refolding,Purification and Activity Assessment Using a ‘Form Invariant’ Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed ‘form invariant’ assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.     

Methods of Preparation of Recombinant Cytokines: IV. Renaturation of Recombinant Human Interleukin-3

Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized. Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions. An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy. It was thereby found that at pH 8.5, which is the optimal value for renaturation, the protein has the secondary structure most close to the native one. The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 × 10⁷ U/mg.     

Purification and characterization of arginine decarboxylase from cucumber (Cucumis sativus) seedlings

A simple, reproducible and rapid protocol for the purification of arginine decarboxylase fromCucumis sativus seedlings has been standardised. The purification steps involved ion-exchange chromatography on diethylaminoethyl-cellulose followed by gel filtration on Sephadex G-l 50. The purified enzyme preparation migrated as a single stainable band on Polyacrylamide gels at both basic and acidic pH, but under denaturing and reducing conditions on sodium dodecyl sulphate-polyacrylamide gels resolved into polypeptides of molecular weight 48,000,44,000 and 15,000. However, in the absence of 2-mercaptoethanol on electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, the enzyme moved as single band with a molecular weight of 150,000. Evidence was obtained to indicate that these three polypeptides were probably derived from a single larger molecular weight enzyme. On storage of the purified protein, the 48,000 species was preferentially degraded to smaller polypeptides. The preliminary data suggested that the 48,000 and 44,000 species shared many common tryptic peptides as revealed by finger printing of the [¹²⁵I ]-labelled protein. The purified enzyme was a glycoprotein and had aK _m of 0.5 mM for arginine. Its activity was stimulated by dithiothrietol and pyridoxal phosphate. EDTA did not inhibit the enzyme activity. Mn²⁺ at 1 mM stimulated arginine decarboxylase activity but was inhibitory at higher concentration     

The SalGI restriction endonuclease. Purification and properties

The type II restriction endonuclease SalGI has been purified to near homogeneity. At least 80% of the protein remaining after the final stage of the preparation is SalGI restriction endonuclease; no contaminating nucleases remain detectable. The principal form of the protein under both native and denaturing conditions is a monomer of M(r) about 29000. The optimal conditions for both enzyme stability and enzyme activity have been determined.     

Partial purification and characterization of a DNA helicase from chloroplasts of Glycine max

A DNA helicase activity was detected in extracts of purified chloroplasts from the SB-1 cell line of Glycine max and partially purified by column chromatography on DEAE cellulose, phosphocellulose, and single-stranded DNA cellulose. The chloroplast helicase has a DNA-dependent ATPase activity, and its strand displacement activity is strictly dependent upon the presence of a nucleoside triphosphate and Mg²⁺ or Mn²⁺. Strand displacement activity does not require a free unannealed single-strand or replication fork-like structure.     

Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell

Background

RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.

Methods

The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.

Results

Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA^Lys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.

Conclusions

The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.

General significance

The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.     

Purification and characterization of laccase II of Aspergillus nidulans

Sexual development in Aspergillus nidulans is a morphogenetic differentiation process triggered by internal and environmental signals. As a first step in analyzing the developmental pathway at the molecular level, laccase II (EC 1.10.3.2), which is specifically expressed in early stages of fruitbodies, was isolated. The enzyme was purified to apparent homogeneity from a mutant strain (SMS1) in which the sexual cycle dominates and the number of cleistothecia is increased tenfold. Laccase II was enriched 560-fold to a specific activity of 892 U (mg protein)^–1. The apparent molecular mass was determined to be 80 kDa under denaturing conditions and to be 100–120 kDa under native conditions. The internal peptide sequences gained from the protein will allow the isolation of the corresponding gene as a first step in determining the key regulators of sexual development. Received: 8 January 1998 / Accepted: 14 April 1998     

Prednisolone enhances aminopeptidase turnover in adult rat small intestine

In adult male rats, fed prednisolone (0.75 mg/kg/day) for 7 days, brush border aminopeptidase activity was increased (P < 0.001) by 106% compared to pair-fed controls. [¹⁴C]Tyrosine was injected intraperitoneally 16 h and [³H]tyrosine 6 h before death. The ³H/¹⁴C ratio was 1.79 ± 0.21 (S.D.) in purified microvillus membranes from treated rats compared to 1.30 ± 0.16 (P < 0.01) in controls. Polyacrylamide gel electrophoresis of brush border membranes under denaturing conditions showed that the increased double-isotope ratio in membranes from treated rats was mainly in the high molecular weight protein subunits (> 80 kDa) Detergent-solubilized aminopeptidase was purified after in vivo labeling by protein A-Sepharose-antiaminopeptidase affinity chromatography. The ³H/¹⁴C ratio in aminopeptidase was 2.42 ± 0.15 (P < 0.05) in treated rats compared to 1.63 ± 0.13 in controls. Over the experimental period steady-state isotope reutilization and protein labeling was demonstrated and there was no isotope metabolism. Total microvillus membrane lipid content was unaffected by prednisolone. We conclude that prednisolone increases brush border aminopeptidase activity by increasing enzyme turnover. Other high molecular weight brush border proteins were similarly affected.     

Endonuclease of high specific activity in a purified mouse interferon preparation

We find an endonuclease of high specific activity in a purified mouse interferon preparation. The interferon was purified from Ehrlich ascites tumor cultures which were induced with Newcastle disease virus. It has a higher specific activity (1.5 × 10⁹ NIH mouse reference standard interferon units/mg protein) than reported for any interferon preparation but is not homogeneous. We do not know if the endonuclease activity is due to a contaminating protein or to interferon. The endonuclease does not degrade in our conditions polyuridylic acid or double stranded reovirus RNA and does not inactivate the tRNA₂^Gln species from $\text{E. coli}$, or tRNA^Val species or polysomes from mouse L cells. Endonuclease in as little as 0.5 ng protein of the interferon preparation degrades μg quantities of messenger RNA from mouse L cells, of encephalomyocarditis virus RNA and of $\text{in vitro}$-synthesized reo-virus messenger RNA at 37° in 1 hour. Further characteristics of the endonuclease and its possible relationship (if any) to interferon remain to be established.     

Analysis of the Effects of Charge Cluster Mutations in Adeno-Associated Virus Rep68 Protein In Vitro

The Rep78 and Rep68 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which are required for viral replication, regulation of AAV promoters, and preferential integration of the AAV genome into a region of human chromosome 19. These proteins bind the hairpin structures formed by the AAV inverted terminal repeat (ITR) origins of replication, make site- and strand-specific endonuclease cuts within the AAV ITRs, and display nucleoside triphosphate-dependent helicase activities. Additionally, several mutant Rep proteins display negative dominance in helicase and/or endonuclease assays when they are mixed with wild-type Rep78 or Rep68, suggesting that multimerization may be required for the helicase and endonuclease functions. Using overlap extension PCR mutagenesis, we introduced mutations within clusters of charged residues throughout the Rep68 moiety of a maltose binding protein-Rep68 fusion protein (MBP-Rep68Δ) expressed in Escherichia coli cells. Several mutations disrupted the endonuclease and helicase activities; however, only one amino-terminal-charge cluster mutant protein (D40A-D42A-D44A) completely lost AAV hairpin DNA binding activity. Charge cluster mutations within two other regions abolished both endonuclease and helicase activities. One region contains a predicted alpha-helical structure (amino acids 371 to 393), and the other contains a putative 3,4 heptad repeat (coiled-coil) structure (amino acids 441 to 483). The defects displayed by these mutant proteins correlated with a weaker association with wild-type Rep68 protein, as measured in coimmunoprecipitation assays. These experiments suggest that these regions of the Rep molecule are involved in Rep oligomerization events critical for both helicase and endonuclease activities.     

Apurinic endonuclease activity from wild-type and repair-deficient mei-9 Drosophila ovaries.

Summary An endonuclease which acts on apurinic (AP) sites in DNA was partially purified from Drosophila ovaries. The enzyme present in the female germ line has a molecular weight of 63 000 daltons, is Mg⁺⁺ dependent, and produces a site upon cleaving depurinated DNA that supports DNA repair synthesis. Although the same characteristics are shared by the enzyme present in the excision-deficient mutant mei-9, specific activity for the AP endonuclease is reduced 98% when compared with that found for its wild-type counterpart. Moreover, cross-reactivity toward an antibody that recognizes the wild-type AP endonuclease protein is reduced roughly 90% for partially purified preparations from mei-9. Mixing experiments between extracts of mei-9 and wild type suggest that the mei-9 structural gene somehow alters or influences the levels of the AP endonuclease protein, but in view of the complex phenotype of this mutant the endonuclease is probably not the product of the gene itself.     

Development of a simple fed-batch process for the high-yield production of recombinant Japanese encephalitis virus protein

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-β-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l⁻¹ and 720 mg l⁻¹ of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.     

Characterization of <Emphasis Type="Italic">Sulfolobus islandicus</Emphasis> rod-shaped virus 2 gp19, a single-strand specific endonuclease

The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg²⁺ for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection.     

DNA binding and pairing activity of OsDmc1, a recombinase from rice

A cloned cDNA corresponding to OsDMC1 from rice anther tissue was expressed in Escherichia coli. The OsDmc1 protein was largely present in the inclusion bodies of the cell lysatE., which was solubilized by 8.0 M urea containing buffeR., purified to homogeneity by Ni-CAM agarose column chromatography, followed by renaturation to its native state through stepwise dialysis against reduced concentrations of urea. The purified protein cross-reacted with anti-yeast Dmc1 antibodies. The binding efficiency observed with circular single-stranded DNA (ssDNA) was similar to that with circular double-stranded DNA (dsDNA). The binding to either DNA showed no ATP dependencE., but required 5–10 mM Mg²⁺ in the presence of ATP. Even though the protein binding to dsDNA was as efficient as it was to ssDNA, the former induced no DNA dependent ATPasE., whereas the binding to ssDNA stimulated a significant level of DNA dependent ATPase activity. OsDmc1–ssDNA complex, with its ATPase proficiency, also mediated renaturation of homologous complementary strands as well as assimilation of single strands into homologous supercoiled duplexes leading to D-loop formation. The D-loop formation was lowered by excess of OsDmc1 protein. This D-loop formation activity was promoted by non-hydrolyzable ATP analog, AMP-PNP and was not observed in absence of ATP or presence of ADP/ATP--S. These properties reflected the classical hallmarks of a recombinase and represented the first biochemical characterization of a plant Dmc1 protein.     

Induced expression of <Emphasis Type="Italic">Eco</Emphasis>RI endonuclease as an active maltose-binding fusion protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Among the numerous bacterial Type II restriction enzymes, EcoRI endonuclease is the most extensively studied and is widely used in recombinant DNA technology. Its heterologous overexpression as recombinant protein has already been studied. However, very limited information concerning its fused product is available thus far. In the present study, the EcoRI restriction endonuclease gene was cloned and expressed as a part of maltose-binding fusion protein under the control of strong inducible tac promoter in TB1 strain of Escherichia coli cells. Transformed cells containing pMALc2X-EcoRI recombinant plasmid were unable to grow under experimental conditions. However, fused EcoRI protein was purified (with the yield of 0.01 mg/l of bacterial culture) by affinity chromatography from E. coli cells induced at the late exponential phase of growth. Restriction quality test revealed that the purified product could restrict a control plasmid DNA in vitro.     

The fission yeast pfh1(+) gene encodes an essential 5' to 3' DNA helicase required for the completion of S-phase

The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease–helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1⁺, encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5′ to 3′ DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the comple tion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.     

Characterization of Recombinant Integrase of Human Immunodeficiency Virus Type 1 (Isolate Bru)

Integration of the human immunodeficiency virus type 1 (HIV-1) DNA into the human genome requires the virusencoded integrase protein. The recombinant integrase protein of HIV-1 (isolate Bru) was prepared by constructing a plasmid based on pET-15b encoding the integrase gene. Integrase of HIV-1 was purified using a bacterial expression system (Escherichia coli). The main kinetic parameters of HIV-1 integrase (K _m = (3.7 ± 0.2)·10^–10 M, k _cat = (1.2 ± 0.3)·10^–7 sec^–1) were determined using an oligonucleotide duplex constructed on the basis of the U5-terminal sequence of proviral HIV-1 DNA as the substrate. Inhibition of integrase by aurintricarbonic acid ([I]₅₀ = 6.3 ± 0.4 M) and dependence of integrase activity on Mg²⁺ and Mn²⁺ concentration were studied.