Protein & Peptide Letters, Volume 12, No. 7, 2005
Contents
Mini-Reviews
Review:
Why is Arginine Effective in Suppressing Aggregation? Pp.613-619
Kohei
Tsumoto, Daisuke Ejima, Yoshiko Kita and Tsutomu Arakawa
Recent
Trends in Protease-Catalyzed Peptide Synthesis Pp.621-629
C.
Lombard, J. Saulnier and J.M. Wallach
Regular
Papers
A
Simple Method for Constructing a Tagged Protein Pp.631-633
C.
Jo, J. Kim and S.A. Jo
Expression,
Renaturation and Functional Analysis of an Excitatory Insect- Specific Toxin
from Scorpion Buthus martensii Karsch Pp.635-638
Chong
Li, Wei Liu, Frank Bossmans, Rong-Huan Zhu, Jan Tytgat and Da-Cheng Wang
Horseradish
Peroxidase Renaturation is Less Efficient at Lower Protein Concentrations Pp.639-643
D.N.
Ermolenko, A.V. Zherdev and B.B. Dzantiev
Characterization
of the Bacteriophage FKMV DNA Ligase Pp.645-648
R.
Lavigne, B. Roucourt, K. Hertveldt and G. Volckaert
Characterization
of Arginine as a Solvent Additive: A Halophilic Enzyme as a Model Protein Pp.649-653
Matsujiro
Ishibashi, Kohei Tsumoto, Daisuke Ejima, Tsutomu Arakawa and Masao Tokunaga
Optimization
of Crystals of an Inhibitory Antibody of Urokinase Plasminogen Activator
Receptor (uPAR) with Hydrogen Peroxide and Low Protein Concentration Pp.655-658
Yongdong
Li, Xiaoli Shi, GrahamParry, Liqing Chen, Jennifer A. Callahan, Andrew P. Mazar
and Mingdong Huang
Expression
and Purification of Soluble Non-Fusion Vasostatin in Escherichia coli Pp.659-661
Xiaoping
Wu, Xiaokun Li, Zhijian Su, Qing Zheng, Hua Xu, Sixian Wu, Ya Feng and Wen Zhao
Protein
Expression, Crystallization and Preliminary X-Ray Crystallographic Studies of
YjbK from Bacillus subtilis Pp.663-664
Xue-Yu
Dai, Yuan Liu, Yu-He Liang, Xiaofeng Zheng, Ming Luo, Lanfen Li and Xiao-Dong
Su
A
Novel Markov Pairwise Protein Sequence Alignment Method for Sequence Comparison Pp.665-669
Xing-Ming
Zhao, Yiu-ming Cheung and De-Shuang Huang
The
Renaturation of Procarboxypeptidase B by Urea Gradient Gel Filtration and Some
Properties of Recombinant Carboxypeptidase B Pp.671-676
Zhang
Xiao-Yan, Li Su-Xia and Yuan Qin-Sheng
Predicted
and Measured Disorder in Peripherin/rds, a Retinal Tetraspanin Pp.677-686
L.M.
Ritter, T. Arakawa and A.F.X. Goldberg
Conformational
Stability of Calreticulin Pp.687-693
Charlotte
S. Jørgensen, Christa Trandum, Nanna Larsen, L. Rebekka Ryder, Michael Gajhede,
Lars K. Skov, Peter Hojrup, Vibeke Barkholt and Gunnar Houen
Purification
and Characterization of a b-Glucuronidase Present During Embryogenesis of the Mollusk
Pomacea sp.
Pp.695-700
Wogelsanger
O. Pereira, Ana K.M. Cruz, Elizabeth M.M. Albuquerque, Elizeu A. Santos,
Adeliana S. Oliveira, Mauricio P. Sales, Fernanda W. Oliveira
New
Derivatives of Picolinic Acid and Nicotinic Acid with Anticonvulsant Activity Pp.701-704
Ryszard
Paruszewski, Marzanna Strupinska, Grazyna Rostafinska-Suchar and James P.
Stables
Crystallization
Reports
Crystallization
and Preliminary Crystallographic Studies of an Antitumour Lectin from the
Edible Mushroom Agrocybe aegerita Pp.705-707
Na
Yang, Yi Liang, Ye Xiang, Ying Zhang, Hui Sun and Da-ChengWang
Crystal
Structure of Non-Fused Glutathione S-Transferase from Schistosoma
japonicum in Complex with Glutathione Pp.709-712
I. Kursula, A.M. Heape and P. Kursula
Crystallization
and Preliminary Crystallographic Analysis of Human Eukaryotic Translation
Initiation Factor 5A (eIF-5A) Pp.713-715
Yuna Sun, Xuemei Li, Beili Wu, Ping Sun and Zihe Rao
Protein
Preparation, Crystallization and Preliminary X-Ray Crystallographic Studies of
Dihydroorotase from Bacillus subtilis Pp.717-719
Yu-He Liang, Xiangyu Liu, Juan Wang, Lanfen Li and Xiao-Dong Su
Abstracts
[Back to top] Review:
Why is Arginine Effective in Suppressing Aggregation?
Kohei
Tsumoto, Daisuke Ejima, Yoshiko Kita and Tsutomu Arakawa
Arginine is finding a wide range of applications in production of proteins. Arginine has been used for many years to assist protein refolding. This effect was ascribed to aggregation suppression by arginine of folding intermediates during protein refolding. Recently, we have observed that arginine facilitates elution of antibodies during Protein-A chromatography and solubilizes insoluble proteins from inclusion bodies, which both can be ascribed to weakening of protein-protein interactions. In order to gain understanding on why arginine is effective in reducing protein-protein interactions and suppressing aggregation, the effects of arginine on stability and solubility of pure proteins have been examined, which showed that arginine is not a protein-stabilizer, but is an aggregation suppressor. However, there is no explanation proposed so far on why arginine suppresses aggregation of proteins. This review addresses such question and then attempts to show differences between arginine and strong denaturants, which are also known as an aggregation suppressor.
[Back to top]
Recent Trends in
Protease-Catalyzed Peptide Synthesis
C.
Lombard, J. Saulnier and J.M. Wallach
Enzymatic peptide syntheses may be either thermodynamically- or kinetically-controlled. The former may be catalyzed by any proteases; the latter is limited to serine and cysteine proteases. This methodology is quite stereospecific and avoids side chain protection but is suffering of some drawbacks. Thus, only two industrial processes are by now developed: the production of aspartame and the conversion of porcine into human insulin. However, recent improvements have been carried out in different directions:
1-Search for proteases with high and/or new P’1 and P1 specificities.
2-Protease engineering to promote synthesis towards hydrolysis and to enlarge specificity.
3-Development of mimetic or "inverse" substrates to limit further hydrolysis of synthesized peptide.
4-Change of the physical state of reactants. Three axes have mainly be explored: solid-solid conversion, use of cross-linked enzyme crystals (CLEC) and enzyme immobilization.
5-Modification of experimental conditions. The principal and recent developments deal with: heterogeneous catalysis, synthesis in low water-containing organic solvents, in ionic liquids or at subzero temperatures.
This review will illustrate these new orientations with examples described in the recent literature.
[Back to top]
A Simple Method for
Constructing a Tagged Protein
C.
Jo, J. Kim and S.A. Jo
We have developed a simple method for preparing a tagged protein by PCR. With this method any protein sequence can be easily tagged. The techniques include three steps of DNA restriction, ligation and PCR. We could obtain a DNA construct containing SUMO-1 gene with His6 tag sequence with high efficiency by the next day.
[Back to top] Expression, Renaturation and Functional Analysis of an
Excitatory Insect- Specific Toxin from Scorpion Buthus martensii Karsch
Chong
Li, Wei Liu, Frank Bossmans, Rong-Huan Zhu, Jan Tytgat and Da-Cheng Wang
The cDNA of BmK IT-AP, an excitatory insect toxin from the scorpion Buthus martensi Karsch that has an analgesic effect on mammalian cells, was expressed in E. coli in the form of an inclusion body. Following denaturation and reduction, the recombinant protein was renatured and purified by liquid chromatography. The authenticity of the recombinant product was confirmed by bioassay and its electrophysiological effect on insect sodium channel.
[Back
to top] Horseradish Peroxidase
Renaturation Is Less Efficient at Lower Protein Concentrations
D.N.
Ermolenko, A.V. Zherdev and B.B. Dzantiev
Renaturation of horseradish peroxidase from guainidine hydrochloride has been studied. Although refolding of the secondary structure was complete, only partial heme incorporation and recovery of enzymatic activity were observed. Heme capturing became less efficient at lower peroxidase concentrations: the refolding yield decreased from 60% at 1 mM to 10% at 0.1 mM concentration of the protein. Probing with conformation-sensitive antibodies indicated structural differences between peroxidase refolded at low concentration and the holo-enzyme.
[Back to top] Characterization of the
Bacteriophage FKMV DNA Ligase
R.
Lavigne, B. Roucourt, K. Hertveldt and G. Volckaert
Gene 17 product (gp17) of the Pseudomonas aeruginosa-infecting bacteriophage fKMV shows in silico similarity to T7 DNA ligase. In a semi-quantitative activity assay, it is shown that gp17 is a functional, ATP-dependent DNA ligase, in spite of some structural differences related to DNA-binding properties). Enzymatic activity of His6-based purified expression product was optimised (4°C at 24h for sticky end double-stranded DNA fragments) and estimated at 0.5 Weiss U/µg.
[Back to top] Characterization of Arginine as a
Solvent Additive: A Halophilic Enzyme as a Model Protein
Matsujiro
Ishibashi, Kohei Tsumoto, Daisuke Ejima, Tsutomu Arakawa and Masao Tokunaga
Arginine suppresses the aggregation of proteins. However, little is known about its mechanism. Here we have used HsNDK (Halobacterium salinarum nucleoside diphosphate kinase) to examine the solvent property of arginine. After exposure to 2 M arginine, HsNDK was diluted to a low salt buffer, resulting in fully active protein. Since unfolded HsNDK cannot refold in such low salt buffer, the observed activity indicates that HsNDK was in the native state in 2 M arginine. Enzyme activity was also examined directly in the presence of arginine, showing that it was active in the presence of 1 M arginine and, to less extent, 2 M arginine. Arginine, however, could not support refolding of heat-denatured HsNDK. HsNDK was stable at 40 ˚C for 19 h incubation in the presence of 1M arginine.
[Back to top] Optimization of Crystals of an
Inhibitory Antibody of Urokinase Plasminogen Activator Receptor (uPAR) with
Hydrogen Peroxide and Low Protein Concentration
Yongdong
Li, Xiaoli Shi, GrahamParry, Liqing Chen, Jennifer A. Callahan, Andrew P. Mazar
and Mingdong Huang
Optimization of protein crystal formation is often a necessary step leading to diffraction-quality crystals to enable collection of a full X-ray data set. Typical protein crystal optimization involves screening different components, e.g., pH, precipitants, and additives of the precipitant solution. Here we present an example using an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) where such procedures did not yield diffracting crystals. In contrast, it was the treatment of the protein with hydrogen peroxide incubation and the protein concentration reduction that were found to be key factors in obtaining diffracting crystals. Final crystals diffracted to 1.75 Å, and belong to orthorhombic P212121 space group with unit cell parameters a=37.162 Å, b=84.474 Å, c=134.030 Å, and contain one molecule of Fab fragment of anti-uro kinase receptor antibody in the asymmetric unit.
[Back to top] Expression and Purification of
Soluble Non-Fusion Vasostatin in Escherichia coli
Xiaoping
Wu, Xiaokun Li, Zhijian Su, Qing Zheng, Hua Xu, Sixian Wu, Ya Feng and Wen Zhao
Vasostatin has previously been expressed in fused form or in inclusion body form in Escherichia coli. Here the protein was expressed in soluble non-fusion form in BL21(DE3)pLysS by IPTG induction. The expression level of vasostatin was about 15% of the total cellular protein. The expressed vasostatin was purified and its biological activity was investigated by an endothelial cell proliferation assay.
[Back to top] Protein
Expression, Crystallization and Preliminary X-Ray Crystallographic Studies of
YjbK from Bacillus subtilis
Xue-Yu
Dai, Yuan Liu, Yu-He Liang, Xiaofeng Zheng, Ming Luo, Lanfen Li and Xiao-Dong
Su
B. subtilis YjbK is a protein with 190 residues of uncharacterized function, it has been annotated by Pfam database as a member of adenylate cyclase family (EC: 4.6.1.1). In order to identify its exact function via structural studies, yjbK gene was amplified from B. subtilis genomic DNA and cloned into expression vector pET21-DEST. The protein was expressed in a soluble form in E. coli and purified to homogeneity. YjbK was crystallized and diffracted to a resolution of 2.0 Å in-house. The crystals belong to P1 space group, with unit cell parameters a=32.38 Å, b=34.69 Å, c=46.02 Å, a=96.560°, b=99.683°, g=111.333°. There is one molecule per asymmetric unit.
[Back to top] A
Novel Markov Pairwise Protein Sequence Alignment Method for Sequence Comparison
The Smith-Waterman (SW) algorithm is a typical technique for local sequence alignment in computational biology. However, the SW algorithm does not consider the local behaviours of the amino acids, which may result in loss of some useful information. Inspired by the success of Markov Edit Distance (MED) method, this paper therefore proposes a novel Markov pairwise protein sequence alignment (MPPSA) method that takes the local context dependencies into consideration. The numerical results have shown its superiority to the SW for pairwise protein sequence comparison.
[Back to top] The Renaturation
of Procarboxypeptidase B by Urea Gradient Gel Filtration and Some Properties of
Recombinant Carboxypeptidase B
Zhang
Xiao-Yan, Li Su-Xia and Yuan Qin-Sheng
A new pro-carboxypeptidase (pCPB) gene was cloned by RT-PCR from SD rat pancreas and its overexpression in Escherichia coli resulted in the formation of inclusion bodies (IBs). The IBs of pCPB were solubilized in 8 M urea and successively refolded by urea gradient gel filtration. Subsequently, the renatured pCPB was digested by trypsin. Recombinant active CPB was obtained by passing through DEAE-FF ion exchange and Sephadex-G100 chromatographic column. Capillary electrophoresis assay showed that the purity of the recombinant CPB (rCPB) exceeded 90%. Further, some properties of rCPB were characterized. The optimum of activity was achieved at pH 7-9. The activity of rCPB was inhibited by typical metal chelating agents (EDTA) and Hg2+, and was activated by Co2+ and heat treatment at 40ºC. The two-dimension electrophoresis map of rCPB showed that the pI value of rCPB was 5.35. UV absorbance spectrum of the enzyme showed that an absorbance maximum was at 277 nm.
[Back to top] Predicted
and Measured Disorder in Peripherin/rds, a Retinal Tetraspanin
L.M.
Ritter, T. Arakawa and A.F.X. Goldberg
Vertebrate photoreceptor outer segment (OS) morphogenesis requires peripherin/rds (P/rds). We have characterized this protein’s C-terminus and present evidence that suggests it is intrinsically disordered. We propose that structural flexibility may underlie the multifunctionality proposed for this domain previously. The extremely short C-termini present in other tetraspanin family members suggest that intrinsic disorder may also play a role for those proteins.
[Back to top] Conformational
Stability of Calreticulin
Charlotte
S. Jørgensen, Christa Trandum, Nanna Larsen, L. Rebekka Ryder, Michael Gajhede,
Lars K. Skov, Peter Hojrup, Vibeke Barkholt and Gunnar Houen
The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (Tm) increased from 31 °C at pH 5 to 51 °C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C-terminal a-helix was of major importance to the conformational stability of calreticulin.
[Back to top] Purification
and Characterization of a b-Glucuronidase Present During Embryogenesis of the Mollusk Pomacea
sp.
Wogelsanger
O. Pereira, Ana K.M. Cruz, Elizabeth M.M. Albuquerque, Elizeu A. Santos,
Adeliana S. Oliveira, Mauricio P. Sales, Fernanda W. Oliveira
A b-glucuronidase was purified from Pomacea sp. eggs by ammonium sulfate fractionation, DEAE-BioGel and Heparin-Sepharose chromatographies. This enzyme showed a Mr 180 kDa, with subunits of 90 kDa. The kinetic parameters were: pH 4.0, temperature 60 0C, Km 2.7 x 10-6 and Vmax 15.3 mM/h, activator Mg+2, and inhibitor: lactone of D-saccharic acid. b-glucuronidase is an exoglucuronidase involved in glycosaminoglycans metabolism with kinetics parameters similar to those found in mammals.
[Back to top] New
Derivatives of Picolinic Acid and Nicotinic Acid with Anticonvulsant Activity
Ryszard
Paruszewski, Marzanna Strupin´ska, Graz•yna Rostafi n´ska-Suchar and James P.
Stables
Previously obtained Pic-BZA is a potent anticonvulsant with low neurotoxicity, but its half-time of action is only about 15 min. In search for equally effective anticonvulsants but with a longer time of action fourteen Pic-BZA analogs were obtained. The compounds were evaluated in the Anticonvulsant Screening Project (ASP) of Antiepileptic Drug Development Program (ADDP) of NIH. Picolinic acid 2-fluorobenzylamide (Pic-2-F-BZA, 7) appeared to be the most effective compound of the series.
[Back to top] Crystallization
and Preliminary Crystallographic Studies of an Antitumour Lectin from the
Edible Mushroom Agrocybe aegerita
Na
Yang, Yi Liang, Ye Xiang, Ying Zhang, Hui Sun and Da-ChengWang
An antitumour lectin named AAL has been purified from the fruiting body of edible mushroom Agrocybe aegerita. In addition to having a distinct bioactivity, AAL shows strong inhibition effects on human and mouse tumour cells. It has been shown that AAL exerts its antitumour effects via apoptosis-induction. AAL and AAL-lactose complex have been crystallized and their diffraction data were collected with resolution of 2.6 Å and 3.0 Å, respectively. Both crystals belong to space group P 6122 with unit cell parameters a = 123.98 Å, b = 123.98 Å, c = 56.86 Å, a= b=90˚, g = 120˚ and a = 123.69 Å, b = 123.69 Å, c = 56.64 Å, a= b=90˚, g = 120˚, respectively.
[Back to top] Crystal Structure of Non-Fused
Glutathione S-Transferase from Schistosoma japonicum in Complex
with Glutathione
I. Kursula, A.M. Heape and P. Kursula
Schistosoma japonicum glutathione S-transferase (SjGST) is a common fusion tag in recombinant protein production, and its 3-dimensional structure has been studied in the context of drug design. We have determined the crystal structure of non-fused SjGST complexed with glutathione, and compare it to complexes between glutathione and SjGST fusion proteins.
[Back to top] Crystallization and Preliminary
Crystallographic Analysis of Human Eukaryotic Translation Initiation Factor 5A
(eIF-5A)
Yuna Sun, Xuemei Li, Beili Wu, Ping Sun and Zihe Rao
Eukaryotic translation initiation factor 5A (eIF-5A) is universally found in all eukaryotic cells. It is the only protein in nature known to contain the unusual amino acid hypusine, a post-translationally modified lysine. Recombinant human eIF-5A was crystallized by the hanging-drop vapor diffusion method. Crystals were grown at 291K using (NH4)2SO4 as precipitant. Diffraction data were obtained to a resolution of 2.7Å from a single frozen crystal belonging to space group C2, with unit-cell parameters a=147.1Å, b=60.4Å, c=76.4Å, b=92.4°. There are more than three molecules per asymmetric unit.
[Back to top] Protein Preparation,
Crystallization and Preliminary X-Ray Crystallographic Studies of
Dihydroorotase from Bacillus subtilis
Yu-He Liang, Xiangyu Liu, Juan Wang, Lanfen Li and Xiao-Dong Su
B. subtilis dihydroorotase is an important enzyme in de novo pyrimidine biosynthesis pathway and encoded by pyrC gene in pyr operon. pyrC was amplified from B. subtilis genomic DNA and cloned into expression vector pET21-DEST. Dihydroorotase was expressed soluble form in E. coli and purified. The protein was crystallized and diffracted to 2.2 Å. The crystal belongs to P212121 space-group, with unit cell parameters a=48.864Å, b=84.99Å, c=203.05Å. There are 2 molecules per asymmetry unit.