[Application of a prediction model in inclusion body refolding.]

Nan Fang Yi Ke Da Xue Xue Bao. 2009 Nov 20;29(11):2156-2160

Authors: Zhang T, Wang JF, Feng YY, Yang Z, Ma L, Wang XN

OBJECTIVE: To establish a prediction method for the refolding of inclusion bodies and classify refolding types of different inclusion bodies directly from their primary structure to improve the efficiency of high throughput refolding process. METHODS: Forty-three recombinant proteins performing important biological functions were expressed in E. coli. The probability of forming inclusion bodies of these proteins was predicted using Harrison's two parameter prediction model based on the proteins' amino acid composition. Subsequently, the proteins from the inclusion bodies were refolded using a double denaturation method that involved washing and denaturation in GdnHCl solution followed by denaturation in Urea solution and refolding through dilution. RESULTS: All the proteins were detected in the form of inclusion bodies using SDS-PAGE method. The proteins were divided into two types according to the results of both solubility prediction and refolding experiments. Fourteen proteins were predicted to have the dependency of soluble expression. The refolding yields of these inclusion bodies were up to 70%. Twenty-nine proteins were predicted to have the high dependency of insoluble expression, and their refolding yields could be higher than 70% and lower than 60%. Comparison of the characteristics between the proteins with high and low refolding yields showed that the theoretical pI was significantly different (P<0.05). CONCLUSIONS: Harrison's two parameter prediction model has the value for potential application in classification of the inclusion bodies and prediction of solubility of proteins refolded from different inclusion bodies. This a novel method enhances the efficiency of high throughput refolding of inclusion bodies, and suggests that the theoretical pI of the proteins is an important parameter in the prediction of refolding yields.

PMID: 19923054 [PubMed - as supplied by publisher]

[Prokaryotic expression, purification of human LINGO-1(aa76-319) and preparation of its polyclonal antibody.]

Nan Fang Yi Ke Da Xue Xue Bao. 2009 Nov 20;29(11):2175-2178

Authors: Lv J, Lu X, Jiang XD, Hu CC, Cai YQ, DU MX, Zou YX, Qin LS

OBJECTIVE: To express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb). METHODS: The 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting. RESULTS: The prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity. CONCLUSION: The fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.

PMID: 19923059 [PubMed - as supplied by publisher]

Chemical Unfolding of Enolase from Saccharomyces cerevisiae Exhibits a Three-State Model.

Protein J. 2009 Nov 19;

Authors: Sánchez-Miguel DS, Romero-Jiménez J, Reyes-López CA, Cabrera-Ávila AL, Carrillo-Ibarra N, Benítez-Cardoza CG

Enolase is a multifunctional protein that participates in glycolysis and gluconeogenesis and can act as a plasminogen receptor on the cell surface of several organisms, among other functions. Despite its participation in a variety of biological and pathophysiological processes, its stability and folding/unfolding reaction have not been fully explored. In this paper we present, the urea and GdnHCl-induced denaturation of enolase studied by means of fluorescence and circular dichroism spectroscopies. We found that enolase unfolds through a highly reversible pathway, populating a stable intermediate species in a range of experimental conditions. The refolding reaction also exhibits an intermediate state that might have a slightly more compact conformation compared to the unfolding intermediate. The thermodynamic parameters associated with the unfolding reaction are presented and discussed.

PMID: 19924403 [PubMed - as supplied by publisher]

Hyperthermophilic archaeal prefoldin shows refolding activity at low temperature.

Biochem Biophys Res Commun. 2009 Nov 14;

Authors: Zako T, Banba S, Sahlan M, Sakono M, Terada N, Yohda M, Maeda M

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself. In this study, we have demonstrated for the first time that a prefoldin from hyper thermophilic archaeon, Pyrococcus horikoshii OT3 (PhPFD), exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures. Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein folding seems to occur through binding and release from PhPFD by virtue of the weak affinity. Our results also imply that that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak.

PMID: 19919829 [PubMed - as supplied by publisher]

Chaperone domains convert prolyl isomerases into generic catalysts of protein folding.

Proc Natl Acad Sci U S A. 2009 Nov 17;

Authors: Jakob RP, Zoldák G, Aumüller T, Schmid FX

The cis/trans isomerization of peptide bonds before proline (prolyl bonds) is a rate-limiting step in many protein folding reactions, and it is used to switch between alternate functional states of folded proteins. Several prolyl isomerases of the FK506-binding protein family, such as trigger factor, SlyD, and FkpA, contain chaperone domains and are assumed to assist protein folding in vivo. The prolyl isomerase activity of FK506-binding proteins strongly depends on the nature of residue Xaa of the Xaa-Pro bond. We confirmed this in assays with a library of tetrapeptides in which position Xaa was occupied by all 20 aa. A high sequence specificity seems inconsistent with a generic function of prolyl isomerases in protein folding. Accordingly, we constructed a library of protein variants with all 20 aa at position Xaa before a rate-limiting cis proline and used it to investigate the performance of trigger factor and SlyD as catalysts of proline-limited folding. The efficiencies of both prolyl isomerases were higher than in the tetrapeptide assays, and, intriguingly, this high activity was almost independent of the nature of the residue before the proline. Apparently, the almost indiscriminate binding of the chaperone domain to the refolding protein chain overrides the inherently high sequence specificity of the prolyl isomerase site. The catalytic performance of these folding enzymes is thus determined by generic substrate recognition at the chaperone domain and efficient transfer to the active site in the prolyl isomerase domain.

PMID: 19920179 [PubMed - as supplied by publisher]

Enzyme-mediated protein refolding.

Chem Commun (Camb). 2009 Dec 14;(46):7197-7199

Authors: Okada J, Maruyama T, Motomura K, Kuroki K, Maenaka K, Sakono M, Goto M

We employed a urease-catalyzed reaction to gradually remove a high concentration of a chaotropic agent (urea) from a denatured protein solution and demonstrated that efficient protein refolding can be achieved by the urease-catalyzed reaction, without large-volume dilution.

PMID: 19921028 [PubMed - as supplied by publisher]

Bacterial production and refolding from inclusion bodies of a "weak" toxin, a disulfide rich protein.

Biochemistry (Mosc). 2009 Oct;74(10):1142-9

Authors: Lyukmanova EN, Shulepko MA, Tikhonov RV, Shenkarev ZO, Paramonov AS, Wulfson AN, Kasheverov IE, Ustich TL, Utkin YN, Arseniev AS, Tsetlin VI, Dolgikh DA, Kirpichnikov MP

The gene for the "weak" toxin of Naja kaouthia venom was expressed in Escherichia coli. "Weak" toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of "weak" toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia "weak" toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain "weak" toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of (125)I-labeled alpha-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (alpha1(2)beta1gammadelta) showed the presence of biological activity of the recombinant "weak" toxin close to the activity of the natural toxin (IC(50) = 4.3 +/- 0.3 and 3.0 +/- 0.5 microM, respectively). The interaction of the recombinant toxin with alpha7 type human neuronal acetylcholine receptor transfected in the GH(4)C(1) cell line also showed the presence of activity close to that of the natural toxin (IC(50) 31 +/- 5.0 and 14.8 +/- 1.3 microM, respectively). The developed bacterial system for production of N. kaouthia venom "weak" toxin was used to obtain (15)N-labeled analog of the neurotoxin.

PMID: 19916927 [PubMed - in process]

Expression, Purification of Herpes Simplex Virus Type 1 UL4 Protein, and Production and Characterization of UL4 Polyclonal Antibody.

J Virol Methods. 2009 Nov 13;

Authors: Pan W, Ren X, Guo H, Ding Q, Zheng AC

The herpes simplex virus type 1 (HSV-1) UL4 protein is a late protein encoded by the UL4 gene. To date, the function of this protein is poorly understood. To aid further investigation of the function of this protein, the UL4 gene was cloned into the vector pET-28a (+) to express His-tagged UL4 protein in Escherichia coli. The recombinant fusion protein was purified from inclusion body by histidine selected nickel affinity chromatography under denaturing conditions. After refolding, the purified recombinant protein was used to produce anti-UL4 polyclonal antibody. Western blot analysis demonstrated that the polyclonal sera could recognize the purified UL4 protein specifically, and in the immunofluorescence assay, the antibody was able to probe the UL4 protein with a punctate staining in HSV-1 infected cells.

PMID: 19917314 [PubMed - as supplied by publisher]

Protein roles in group I intron RNA folding: The tyrosyl-tRNA synthetase CYT-18 stabilizes the native state relative to a long-lived misfolded structure without compromising folding kinetics.

J Mol Biol. 2009 Nov 10;

Authors: Chadee AB, Bhaskaran H, Russell R

The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P5abc functionally. In addition to stabilizing structure generally, P5abc stabilizes the native conformation of the Tetrahymena group I intron relative to a globally-similar misfolded conformation, which has only local differences within the core and is populated significantly at equilibrium by a ribozyme variant lacking P5abc (E(DeltaP5abc)). Here we show that CYT-18 specifically promotes formation of the native group I intron core from this misfolded conformation. Catalytic activity assays demonstrate that CYT-18 shifts the equilibrium of E(DeltaP5abc) toward the native state by at least 35-fold, and binding assays suggest an even larger effect. Thus, like P5abc, CYT-18 preferentially recognizes the native core, despite the global similarity of the misfolded core and despite forming crudely similar complexes, as revealed by DMS footprinting. Interestingly, the effects of CYT-18 and P5abc on folding kinetics differ. Whereas P5abc inhibits refolding of the misfolded conformation by forming peripheral contacts that must break during refolding, CYT-18 does not display analogous inhibition, most likely because it relies to a greater extent on direct interactions with the core. Although CYT-18 does not encounter this RNA in vivo, our results suggest that it stabilizes its cognate group I introns relative to analogous misfolded intermediates. By specifically recognizing native structure, CYT-18 may also interact with earlier folding intermediates to avoid RNA misfolding or to trap native structure as it forms. More generally, our results highlight the ability of a protein cofactor to stabilize a functional RNA structure specifically without incurring associated costs in RNA folding kinetics.

PMID: 19913030 [PubMed - as supplied by publisher]

Refolding dynamics of stretched biopolymers upon force quench.

Proc Natl Acad Sci U S A. 2009 Nov 13;

Authors: Hyeon C, Morrison G, Pincus DL, Thirumalai D

Single-molecule force spectroscopy methods can be used to generate folding trajectories of biopolymers from arbitrary regions of the folding landscape. We illustrate the complexity of the folding kinetics and generic aspects of the collapse of RNA and proteins upon force quench by using simulations of an RNA hairpin and theory based on the de Gennes model for homopolymer collapse. The folding time, tau(F), depends asymmetrically on delta f(S) = f(S) - f(m) and delta f(Q) = f(m) - f(Q) where f(S) (f(Q)) is the stretch (quench) force and f(m) is the transition midforce of the RNA hairpin. In accord with experiments, the relaxation kinetics of the molecular extension, R(t), occurs in three stages: A rapid initial decrease in the extension is followed by a plateau and finally, an abrupt reduction in R(t) occurs as the native state is approached. The duration of the plateau increases as lambda =tau(Q)/tau(F) decreases (where tau(Q) is the time in which the force is reduced from f(S) to f(Q)). Variations in the mechanisms of force-quench relaxation as lambda is altered are reflected in the experimentally measurable time-dependent entropy, which is computed directly from the folding trajectories. An analytical solution of the de Gennes model under tension reproduces the multistage stage kinetics in R(t). The prediction that the initial stages of collapse should also be a generic feature of polymers is validated by simulation of the kinetics of toroid (globule) formation in semiflexible (flexible) homopolymers in poor solvents upon quenching the force from a fully stretched state. Our findings give a unified explanation for multiple disparate experimental observations of protein folding.

PMID: 19915145 [PubMed - as supplied by publisher]

Refolding of an unstable lysozyme by gradient removal of a solubilizer and gradient addition of a stabilizer.

J Biochem. 2009 Nov 11;

Authors: Kohyama K, Matsumoto T, Imoto T

Previously we formally established an effective refolding procedure for a protein by gradient removal of a solubilizer such as urea [Maeda et al. (1995) Effective renaturation of reduced lysozyme by gentle removal of urea. Protein Eng. 8, 201-205]. However, this procedure was less effective for unstable proteins. We developed here an excellent method to add protein stabilizer so as to get reasonable amounts of folded protein under the concentration of solubilizer where the unstable protein does not form aggregate. We examined many stabilizers and found that 60% of a concentrated (2.5 mg/ml) unstable protein can be refolded using 40% glycerol as the best stabilizer. This procedure can be widely applicable for the refolding of unstable proteins.

PMID: 19906826 [PubMed - as supplied by publisher]

Mutations in the ectodomain of the Newcastle Disease Virus Fusion protein confers hemagglutinin-neuraminidase independent phenotype.

J Virol. 2009 Nov 11;

Authors: Ayllón J, Villar E, Muñoz-Barroso I

The entry of enveloped viruses into host cells is preceded by membrane fusion, which in paramyxoviruses is triggered by the fusion (F) protein. Refolding of the F protein from a metastable conformation to a highly stable post-fusion form is critical for the promotion of fusion although the mechanism is still not well understood. Here we examined the effects of mutations of individual residues of the F protein of Newcastle Disease Virus located at critical regions of the protein, such as the C-terminus of the N-terminal heptad repeat (HRA) and the N-terminus of the C-terminal heptad repeat (HRB). Seven of the mutants were expressed at the cell surface, showing differences in antibody reactivity in comparison with the F wild-type. Mutants N211A, L461A, I463A and I463F showed a hyperfusogenic phenotype both in syncitium and in dye-transfer assays. The four mutants promoted fusion more efficiently at lower temperatures than the wild type, meaning probably lower energy requirements for activation. Moreover, mutants N211A, I463A and I463F exhibited haemagglutinin-neuraminidase(HN)-independent activity when influenza haemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.

PMID: 19906934 [PubMed - as supplied by publisher]

Residues in the Heptad Repeat A Region of the Fusion (F) Protein Modulate the Virulence of Sendai Virus in Mice.

J Virol. 2009 Nov 11;

Authors: Luque LE, Bridges OA, Mason JN, Boyd KL, Portner A, Russell CJ

While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion), yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs was similar for wild-type and F-L179V viruses. After approximately one week of infection, the clearance of F-L179V virus was delayed and more extensive interstitial inflammation and necrosis was observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.

PMID: 19906935 [PubMed - as supplied by publisher]

Halophilic beta-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1alpha and human neutrophil alpha-defensin.

Appl Microbiol Biotechnol. 2009 Nov 10;

Authors: Tokunaga H, Saito S, Sakai K, Yamaguchi R, Katsuyama I, Arakawa T, Onozaki K, Arakawa T, Tokunaga M

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. beta-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several "difficult-to-express" proteins as a BLA fusion protein and verified biological activities of human interleukin 1alpha and human neutrophil alpha-defensin, HNP-1.

PMID: 19902204 [PubMed - as supplied by publisher]

Refolding solubilized inclusion body proteins.

Methods Enzymol. 2009;463:259-82

Authors: Burgess RR

The vast majority of protein purification is now done with cloned, recombinant proteins expressed in a suitable host. The predominant host is Escherichia coli. Many, if not most, expressed proteins are found in an insoluble form called an inclusion body (IB). Since the target protein is often relatively pure in a washed IB, the challenge is not so much to purify the target, but rather to solubilize an IB and refold the protein into its native structure, regaining full biological activity. While many of the operations of this process are quite general (expression, cell disruption, IB isolation and washing, and IB solubilization), the precise conditions that give efficient refolding differ for each protein. This chapter describes the main techniques and strategies for achieving successful refolding.

PMID: 19892177 [PubMed - in process]

Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus.

Protein Expr Purif. 2009 Oct 31;

Authors: Kumar GS, Sarma SP

BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome b(5) fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage by E.coli. High expression levels of the fusion protein enabled facile purification of this peptide. The presence of disulfide bonded isomers, occurring as distinctly populated states even in the fusion protein, were separated by gel filtration chromatography. The target peptide was liberated from the host protein by Tev protease cleavage and subsequent purification was achieved using RP-HPLC methods. Reverse phase HPLC clearly showed the presence of at least two isomeric forms of the peptide that were significantly populated. The oxidative folding of BTK-2 was achieved under ambient conditions during the course of purification. Structural characterization of the two forms, by solution homonuclear and heteronuclear NMR methods, has shown that these two forms exhibit significantly different structural properties, and represent the natively folded and a "misfolded" form of the peptide. The formation of properly folded BTK-2 as a major fraction without the use of in vitro oxidative refolding methods clearly indicate the versatility of the Cytochrome b(5) fusion protein system for the efficient production of peptides for high resolution NMR studies.

PMID: 19887110 [PubMed - as supplied by publisher]

Growth Inhibition of Human Melanoma Cells by a Recombinant Arginine Deiminase Expressed in Escherichia coli.

J Vet Med Sci. 2009 Oct;71(10):1343-7

Authors: Kozai M, Sasamori E, Fujihara M, Yamashita T, Taira H, Harasawa R

We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361.

PMID: 19887741 [PubMed - in process]

Chemical shift assignments for human apurinic/apyrimidinic endonuclease 1.

Biomol NMR Assign. 2009 Nov 4;

Authors: Manvilla BA, Varney KM, Drohat AC

Apurinic/apyrimidinic endonuclease 1 (APE1 or Ref-1) is the major enzyme in mammals for processing abasic sites in DNA. These cytotoxic and mutagenic lesions arise via spontaneous rupture of the base-sugar bond or the removal of damaged bases by a DNA glycosylase. APE1 cleaves the DNA backbone 5' to an abasic site, giving a 3'-OH primer for repair synthesis, and mediates other key repair activities. The DNA repair functions are essential for embryogenesis and cell viability. APE1-deficient cells are hypersensitive to DNA-damaging agents, and APE1 is considered an attractive target for inhibitors that could potentially enhance the efficacy of some anti-cancer agents. To enable an important new method for studying the structure, dynamics, catalytic mechanism, and inhibition of APE1, we assigned the chemical shifts (backbone and (13)C(beta)) of APE1 residues 39-318. We also report a protocol for refolding APE1, which was essential for achieving complete exchange of backbone amide sites for the perdeuterated protein.

PMID: 19888678 [PubMed - as supplied by publisher]

Secondary Structure of the Mature Ex Virio Moloney Murine Leukemia Virus Genomic RNA Dimerization Domain.

J Virol. 2009 Nov 4;

Authors: Gherghe C, Leonard CW, Gorelick RJ, Weeks KM

Retroviral genomes are dimeric, comprised of two sense strand RNAs linked at their 5' ends by non-covalent base pairing and tertiary interactions. Viral maturation involves large-scale morphologic changes in viral proteins and in genomic RNA dimer structures to yield infectious virions. Structural studies have largely focused on simplified in vitro models of genomic RNA dimers even though the relationship between these models and authentic viral RNA is unknown. We evaluate the secondary structure of the minimal dimerization domain in genomes isolated from Moloney murine leukemia virions using a quantitative and single nucleotide resolution RNA structure analysis technology (SHAPE). Results are consistent with an architecture in which the RNA dimer is stabilized by four primary interactions involving two sets of intermolecular base pairs and two loop-loop interactions. The dimerization domain can independently direct its own folding since heating and refolding reproduces the same structure as visualized in genomic RNA isolated from virions. Authentic ex virio RNA has a SHAPE reactivity profile similar to that of a simplified-transcript dimer generated in vitro, with the important exception of a region that appears to form a compact stem-loop only in the virion-isolated RNA. Finally, we analyze the conformational changes that accompany folding of monomers into dimers in vitro. These experiments support well-defined structural models for an authentic dimerization domain and also emphasize that many features of mature genomic RNA dimers can be reproduced in vitro using properly designed, simplified RNAs.

PMID: 19889760 [PubMed - as supplied by publisher]

Related Articles

Production of recombinant proteins in E. coli using an N-terminal tag derived from sortase.

Protein Expr Purif. 2009 Oct 30;

Authors: Caswell J, Snoddy P, McMeel D, Buick RJ, Scott CJ

The use of Escherichia coli protein expression systems have many benefits, including the ease of propagation, amounts of protein that can be generated and cost. However, this host has some drawbacks due to difficulties in the production of soluble foreign proteins with their alternate codon usage bias, reductive cytoplasmic environment and lack of complex post-translational modifications. We have designed a novel fusion protein tag derived from the sequence of sortase (SrtA) which we have named Solubility 'eNhancing' Ubiquitous Tag (SNUT). Here we demonstrate its application and effectiveness as an N-terminal fusion tag for the expression and purification of proteins that could not be effectively produced with other tags. We show this tag can be utilized for the purification of proteins through both native and refolding immobilized metal ion chromatography and in combination with an anti-SNUT monoclonal antibody, can also be used as a detection tag. This tag may prove useful in circumventing expression and purification issues with the production of proteins in bacterial expression hosts.

PMID: 19883766 [PubMed - as supplied by publisher]