Cytokine-Mediated Inhibition of Fibrillar Amyloid-{beta} Peptide Degradation by Human Mononuclear Phagocytes.

J Immunol. 2008 Sep 15;181(6):3877-86

Authors: Yamamoto M, Kiyota T, Walsh SM, Liu J, Kipnis J, Ikezu T

Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-beta peptides (Abeta) in brain. Although Abeta uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Abeta degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Abeta using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer (125)I-Abeta40 and Abeta42. Initial uptake of fibrillar Abeta40 and Abeta42 was 40% and its degradation was saturated by 120 h in both MDM and microglia, compared with an initial uptake of oligomeric Abeta less than 0.5% and saturation of degradation within 24 h. IFN-gamma increased the intracellular retention of fibrillar Abeta40 and Abeta42 by inhibiting degradation, whereas IL-4, IL-10, and TGF-beta1, but not IL-13 and IL-27, enhanced degradation. Fibrillar Abeta degradation in MDM is sensitive to lysosomal and insulin degrading enzyme inhibitors but insensitive to proteasomal and neprilysin inhibitors. IFN-gamma and TNF-alpha directly reduced the expression of insulin degrading enzyme and chaperone molecules (heat shock protein 70 and heat shock cognate protein 70), which are involved in refolding of aggregated proteins. Coculture of MDM with activated, but not naive T cells, suppressed Abeta degradation in MDM, which was partially blocked by a combination of neutralizing Abs against proinflammatory cytokines. These data suggest that proinflammatory cytokines suppress Abeta degradation in MDM, whereas select anti-inflammatory and regulatory cytokines antagonize these effects.

PMID: 18768842 [PubMed - in process]

Transport functions dominate the SAR11 metaproteome at low-nutrient extremes in the Sargasso Sea.

ISME J. 2008 Sep 4;

Authors: Sowell SM, Wilhelm LJ, Norbeck AD, Lipton MS, Nicora CD, Barofsky DF, Carlson CA, Smith RD, Giovanonni SJ

The northwestern Sargasso Sea undergoes annual cycles of productivity with increased production in spring corresponding to periods of upwelling, and oligotrophy in summer and autumn, when the water column becomes highly stratified. The biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorus and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary liquid chromatography (LC)-tandem mass spectrometry to detect microbial proteins in surface samples collected in September 2005. A total of 2215 peptides that mapped to 236 SAR11 proteins, 1911 peptides that mapped to 402 Prochlorococcus proteins and 2407 peptides that mapped to 404 Synechococcus proteins were detected. Mass spectra from SAR11 periplasmic substrate-binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars and spermidine. Proteins implicated in the prevention of oxidative damage and protein refolding were also abundant. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme, but nutrient flux is sufficient to sustain microbial community activity.The ISME Journal advance online publication, 4 September 2008; doi:10.1038/ismej.2008.83.

PMID: 18769456 [PubMed - as supplied by publisher]

Purification, crystallization and preliminary X-ray analysis of human mannose-binding lectin-associated serine protease-1 (MASP-1) catalytic region.

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Sep 1;64(Pt 9):781-4

Authors: Dobó J, Harmat V, Sebestyén E, Beinrohr L, Závodszky P, Gál P

MASP-1, a multidomain serine protease, is a component of the lectin pathway of complement. Its precise function is unknown, although it seems to enhance the complement-activating capacity of MASP-2, a related enzyme. MASP-1 has also been implicated as playing a role in blood coagulation. It is mostly found associated with mannose-binding lectin (MBL) and ficolins. Early attempts to crystallize MASP-1 failed because of the inhomogeneity of the purified material. MASP-1 was shown by acidic nondenaturing PAGE to be composed of differently charged species, which are most likely to be the products of deamidation occurring during the refolding procedure. Sequential cation-exchange and anion-exchange chromatography resulted in a homogeneous material, which was successfully crystallized. The best crystal diffracted to 2.55 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 68.4, b = 70.4, c = 121.4 A. The crystal structure of MASP-1 may help in understanding the function of this mysterious serine protease.

PMID: 18765903 [PubMed - in process]

Simplified recombinant plasmin: Production and functional comparison of a novel thrombolytic molecule with plasma-derived plasmin.

Thromb Haemost. 2008 Sep;100(3):413-9

Authors: Hunt JA, Petteway SR, Scuderi P, Novokhatny V

A simplified and fully functional deletion mutant of plasminogen was created in which the middle portion of the molecule was removed, resulting in kringle 1 attachment to the serine protease domain. This recombinant plasminogen deletion mutant, Delta(K2-K5)Pg, was produced in the form of inclusion bodies at the yield of up to 200 mg/l in an Escherichia coli T7 expression system. Following protein refolding and purification on lysine-Sepharose, the conversion of the recombinant molecule Delta(K2-K5)Pg to the active enzyme mutant Delta(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied. Properties of Delta(K2-K5)Pg were similar to native, human plasma-derived plasminogen. Delta(K2-K5)Pg effectively bound epsilon-aminocaproic acid (K(d) = 11.3 +/- 2.3 muM) and fibrin (C(50) approximately 0.3 muM). The plasminogen activators streptokinase, urokinase, and tissue plasminogen activator effectively converted the recombinant zymogen Delta(K2-K5)Pg to the active recombinant enzyme, Delta(K2-K5)Pm. Additionally, Delta[K2-K5]Pm was rapidly inhibited by alpha(2)-antiplasmin (1.1 +/- 0.1 x 10(7) M(-1)s(-1)) and alpha(2)-macroglobulin (7.6 +/- 0.6 x 10(5) M(-1)s(-1)). In an in-vitro model, Delta(K2-K5)Pm demonstrated fibrinolytic potency comparable to human plasma-derived plasmin. Because of their unique biochemistry, including fibrin-binding properties and rapid inhibition by alpha(2)-antiplasmin, both native plasmin and a simplified deletion mutant of plasmin are potentially safe and effective direct thrombolytic agents for various thrombotic conditions. Further studies evaluating the in-vivo pharmacologic safety and clinical efficacy of this simplified plasmin (i.e. Delta[K2-K5]Pm) are warranted.

PMID: 18766256 [PubMed - in process]

MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, Methanothermobacter thermoautotrophicum involving in stress response.

Cell Stress Chaperones. 2008;13(2):239-46

Authors: Ding X, Lv ZM, Zhao Y, Min H, Yang WJ

MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50 degrees C) and high (70 degrees C) growth temperatures than under the optimal growth temperature for the organism (65 degrees C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4 degrees C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0 degrees C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.

PMID: 18759006 [PubMed - in process]

The thermodynamic stability of insulin disulfides is not affected by the C-domain of insulin-like growth factor 1.

Sci China C Life Sci. 2002 Jun;45(3):245-50

Authors: Guo Z, Feng Y

Both Insulin and insulin-like growth factor 1 are members of insulin superfamily. They share homologous primary and tertiary structure as well as weakly overlapping biological activity. However, their folding behavior is different: insulin and its recombinant precursor (PIP) fold into one unique tertiary structure, while IGF-1 folds into two disulfides isomers with similar thermodynamic stability. To elucidate the molecular mechanism of their different folding behavior, we prepared a singlechain hybrid of insulin and IGF-1, [B10Glu]Ins/IGF-1(C), and studied its folding behavior compared with that of PIP and IGF-1. We also separated a major non-native disulfides isomer of the hybrid and studied its refolding. The data showed that the C-domain of IGF-1 did not affect the folding thermodynamics of insulin, that is, the primary structure of the hybrid encoded only one thermodynamically stable disulfides linkage. However, the folding kinetics of insulin was affected by the C-domain of IGF-1.

PMID: 18759047 [PubMed - in process]

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Characterization of Ser73 in Arabidopsis thaliana Glutathione S-transferase zeta class.

J Genet Genomics. 2008 Aug;35(8):507-12

Authors: Tao S, Chen X, Liu J, Ming M, Chong N, Chen D

Glutathione S-transferases (GSTs) are ubiquitous detoxifying superfamily enzymes. The zeta class GST from Arabidopsis thaliana (AtGSTZ) can efficiently degrade dichloroacetic acid (DCA), which is a common carcinogenic contaminant in drinking water. Ser73 in AtGSTZ is a conserved residue at Glutathione binding site (G-site). Compared with the equivalent residues in other GSTs, the catalytic and structural properties of Ser73 were poorly investigated. In this article, site-saturation mutagenesis was performed to characterize the detailed role of Ser73. The DCA dechlorinating (DCA-DC) activity showed that most of the mutants had less than 3% of the wild-type activity, except S73T and S73A showing 43.48% and 21.62% of the wild-type activity, respectively, indicating that position 73 in AtGSTZ showed low mutational substitutability. Kinetic experiments revealed that mutants S73T, S73A, and S73G showed low binding affinity and catalytic efficiency toward DCA, 1.8-, 3.1-, and 10.7-fold increases in K(m)(DCA) values and 4.0-, 9.6-, and 34.1-fold decreases in K(cat-)(DCA)/K(m)(DCA) values, respectively, compared to the wild type. Thermostability and refolding experiments showed that the wild type maintained more thermostability and recovered activity. These results demonstrated the important role of Ser73 in catalytic activity and structural stability of the enzyme. Such properties of Ser73 could be particularly crucial to the molecular evolution of AtGSTZ and might, therefore, help explain why Ser73 is conserved in all GSTs. This conclusion might provide insights into the directed evolution of the DCA-DC activity of AtGSTZ.

PMID: 18721788 [PubMed - in process]

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Structure-based analysis reveals hydration changes induced by arginine hydrochloride.

Biophys Chem. 2008 Aug 9;

Authors: Nakakido M, Tanaka Y, Mitsuhori M, Kudou M, Ejima D, Arakawa T, Tsumoto K

Arginine hydrochloride has been used to suppress protein aggregation during refolding and in various other applications. We investigated the structure of hen egg-white lysozyme (HEL) and solvent molecules in arginine hydrochloride solution by X-ray crystallography. Neither the backbone nor side-chain structure of HEL was altered by the presence of arginine hydrochloride. In addition, no stably bound arginine molecules were observed. The number of hydration water molecules, however, changed with the arginine hydrochloride concentration. We suggest that arginine hydrochloride suppresses protein aggregation by altering the hydration structure and the transient binding of arginine molecules that could not be observed.

PMID: 18725174 [PubMed - as supplied by publisher]

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Simultaneous refolding and purification of a recombinant lipase with an intein tag by affinity precipitation with chitosan.

Biochim Biophys Acta. 2008 Aug 6;

Authors: Singh PK, Gupta MN

A strategy for simultaneous purification and refolding of proteins overexpressed with an intein tag is described. A recombinant lipase overexpressed in Escherichia coli ER2566 with the intein tag and obtained as inclusion bodies was solubilized in buffer containing 8 M urea or cetyltrimethylammonium bromide. The solubilized lipase was precipitated with chitosan and the affinity complex of the polymer with the fusion protein was obtained. The intein tag was cleaved with dithiothreitol and the refolded lipase was obtained in active form. Activity recovery of 80% was observed and the enzyme had a specific activity of 2965 units/mg. The purified lipase showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purity and activity recovery were comparable with that of the preparation obtained by using the commercial kit which utilizes chromatography on chitin beads. The purified and refolded lipase was characterized by fluorescence and CD spectroscopy.

PMID: 18725331 [PubMed - as supplied by publisher]

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Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay.

Photochem Photobiol Sci. 2008 Sep;7(9):1025-31

Authors: Borisova VV, Frank LA, Markova SV, Burakova LP, Vysotski ES

The recombinant coelenterazine-dependent luciferases (isoforms MLuc164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. coli cells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc39) was obtained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free coelenterazine, but also by Ca(2+)-dependent coelenterazine-binding protein (CBP) of Renilla muelleri on Ca(2+) addition. The use of CBP as a "substrate" provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35-40 % of the initial activity. The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca(2+)-regulated photoprotein obelin and the Metridia luciferase.

PMID: 18754048 [PubMed - in process]

Expression of human transforming growth factor beta1 inE. coli (I) : Direct expression in cytoplasm.

Sci China C Life Sci. 1998 Oct;41(5):548-54

Authors: Long J, Wang H, Liu L, Wang F, Zhou T

PCR technique is used to amplify the mature peptide gene of human transforming growth factor pl (hTGFbeta1); the gene is verified by full-length sequence analysis. In DHSa/pBV220 expression system, hTGFbeta1 attains expression in the cytoplasm ofE. coli up to 16%. The recombinant protein is proved to be the monomer of hTGFPl by N-terminal amino acids analysis and immunoblotting. After refolding of the monomer proteinin vitm in glutathione system or CHPAS/DMSO system, the dimeric protein accumulates to 30% in the refolding mixture. The recombinant protein is purified to homogeneity on silver staining, and is shown to have strong biological activity from MTT bioassay on MvlLu cells.

PMID: 18726238 [PubMed - in process]

Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity.

Sci China C Life Sci. 2000 Jun;43(3):232-8

Authors: Yan X, Tang J, Wu X, Wang F, Li J, Yang D

Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

PMID: 18726377 [PubMed - in process]

Peptide and protein binding in the axial channel of Hsp104: insights into the mechanism of protein unfolding.

J Biol Chem. 2008 Aug 28;

Authors: Lum R, Niggemann M, Glover JR

The AAA+ molecular chaperone Hsp104 mediates the extraction of proteins from aggregates by unfolding and threading them through its axial channel in an ATP-driven process. An Hsp104-binding peptide selected from solid phase arrays enhanced the refolding of a firefly luciferase-peptide fusion protein. Analysis of peptide binding using tryptophan fluorescence revealed two distinct binding sites, one in each AAA+ module of Hsp104. As a further indication of the relevance of peptide binding to Hsp104 mechanism, we found that it competes with the binding of a model unfolded protein, reduced carboxymethylated alpha-lactalbumin. Inactivation of the pore loops in either AAA+ module prevented stable peptide and protein binding. However, when the loop in the first AAA+ was inactivated, stimulation of the ATPase turnover in second AAA+ module of this mutant was abolished. Drawing on these data, we propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA+ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA+ module.

PMID: 18755692 [PubMed - as supplied by publisher]

Disulfide Bond Bridged Divalent Antibody-Toxin, (Fab-PE38fl)2, with the Toxin PE38 Fused to the Light Chain.

J Microbiol Biotechnol. 2008 Aug;18(8):1475-81

Authors: Won J, Choe M

B3 antibody specifically binds the LewisY-related carbohydrate antigen of many carcinomas, and it is used as a model antibody in this study. In a previous study, the Fab fragment of the antibody was fused to a 38 kDa truncated form of Pseudomonas exotoxin A, PE38, to make Fab- PE38, where PE38 is fused to the Fd fragment of the Fab domain. This parent monomer molecule, Fab-PE38, had no cysteine in the hinge region, and it could not make a disulfide bond to form a disulfide bond bridged homodimer [7]. In this study, we constructed three different kinds of divalent Fab-toxin fusion homodimers where the toxin is fused to the light chain of Fab, (Fab-PE38fl)2. In addition to the PE38 toxin fused to the light chain, these three molecules have different hinge sequences h1, h2, and h3 making Fabh1-, Fabh2-, and Fabh3-PE38fl monomers, respectively. These hinges contain only one cysteine on different positions of the hinge sequence. The disulfide bond between the hinge region of two monomers forms homodimers (Fabh1-PE38fl)2, (Fabh2-PE38fl)2, and (Fabh3- PE38fl)2. The refolding yields of these dimers were 5- 16-fold higher than a previously constructed dimer where the PE38 was fused to the Fd fragment (Fabh1-PE38)2 [8]. Our data suggest that the steric repulsion between the two PE38s in (Fabh1-PE38)2 during disulfide bridge formation is relieved by fusing it at the end of the light chain. The best cytotoxicity value of these dimers showed about 2.5-fold higher on an MCF7 cell line than that of the monovalent reference molecule in ng/ml scale, which is 15-fold higher in pM scale.

PMID: 18756111 [PubMed - in process]

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High-yield expression in E. coli and refolding of the bZIP domain of activating transcription factor 5.

Protein Expr Purif. 2008 Aug 3;

Authors: Ciaccio NA, Moreno ML, Bauer RL, Laurence JS

Activating transcription factor 5 (ATF5) recently has been demonstrated to play a critical role in promoting the survival of human glioblastoma cells. Interference with the function of ATF5 in an in vivo rat model caused glioma cell death in primary tumors but did not affect the status of normal cells surrounding the tumor, suggesting ATF5 may prove an ideal target for anti-cancer therapy. In order to examine ATF5 as a pharmaceutical target, the protein must be produced and purified to sufficient quantity to begin analyses. Here, a procedure for expressing and refolding the bZIP domain of ATF5 in sufficient yield and final concentration to permit assay development and structural characterization of this target using solution NMR is reported. Two-dimensional NMR and circular dichroism analyses indicate the protein exists in the partially alpha-helical, monomeric x-form conformation with only a small fraction of ATF5 participating in formation of higher-order structure, presumably coiled-coil homodimerization. Despite the persistence of monomers in solution even at high concentration, an electrophoretic mobility shift assay showed that ATF5 is able to bind to the cAMP response element (CRE) DNA motif. Polyacrylamide gel electrophoresis and mass spectrometry were used to confirm that ATF5 can participate in homodimer formation and that this dimerization is mediated by disulfide bond formation.

PMID: 18718539 [PubMed - as supplied by publisher]

Small heat shock protein activity is regulated by variable oligomeric substructure.

J Biol Chem. 2008 Aug 19;

Authors: Benesch JL, Ayoub M, Robinson CV, Aquilina JA

The alpha-crystallins are members of the small heat shock protein (sHSP) family of molecular chaperones which have evolved to minimize intracellular protein aggregation, however they are also implicated in a number of protein deposition diseases. In this study we have employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone to adjuvant of aggregation. We have replicated the oligomeric rearrangements observed for in vivo disease-related modifications, without altering the protein sequence, by refolding the alpha-crystallins in vitro. This refolding results in a loss of dimeric substructure concomitant with an augmentation of substrate affinity. We show that packaging of sHSPs into dimeric units is used to control the level of chaperone function by regulating the exposure of hydrophobic surfaces. We propose that a bias toward monomeric substructure is responsible for the aberrant chaperone behaviour associated with the alpha-crystallins in protein deposition diseases.

PMID: 18713743 [PubMed - as supplied by publisher]

Dictyostelium discoideum--a promising expression system for the production of eukaryotic proteins.

FASEB J. 2008 Aug 19;

Authors: Arya R, Bhattacharya A, Saini KS

In general, four different expression systems, namely, bacterial, yeast, baculovirus, and mammalian, are widely used for the overproduction of biochemical enzymes and therapeutic proteins. Clearly, bacterial expression systems offer ease of maneuverability with respect to large-scale production of recombinant proteins, while, a baculovirus expression system ensures proper protein modifications, processing, and refolding of complex proteins. Despite these advantages, mammalian cells remain the preferred host for many eukaryotic proteins of pharmaceutical importance, particularly, those requiring post-translational modifications. Recently, the single-celled slime mold, Dictyostelium discoideum (Dd), has emerged as a promising eukaryotic host for the expression of a variety of heterologous recombinant eukaryotic proteins. This organism possesses the complex cellular machinery required for orchestrating post-translational modifications similar to the one observed in higher eukaryotes. This review summarizes the advantages and disadvantages of Dictyostelium as an alternate system compared to other well-established expression systems. The key lessons learned from the expression of human recombinant proteins in this system are reviewed. Also, the strengths, weaknesses, and challenges associated with industrial-scale production of proteins in Dd expression system are discussed.-Arya, R., Bhattacharya, A., Saini, K. S. Dictyostelium discoideum-a promising expression system for the production of eukaryotic proteins.

PMID: 18714070 [PubMed - as supplied by publisher]

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Kinetic Coupling of Folding and Prolyl Isomerization of beta(2)-Microglobulin Studied by Mutational Analysis.

J Mol Biol. 2008 Aug 7;

Authors: Sakata M, Chatani E, Kameda A, Sakurai K, Naiki H, Goto Y

beta(2)-Microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of beta2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of beta2-m, we studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V beta2-ms) as well as the wild-type beta2-m. W39 beta2-m is a triple mutant in which both of the authentic Trp residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95/F). W39 beta2-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V beta2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic coupling of unfolding/refolding and prolyl isomerization, which has tended to be neglected in recent studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance.

PMID: 18708068 [PubMed - as supplied by publisher]

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Recombinant human SMOCs produced by in vitro refolding: Calcium-binding properties and interactions with serum proteins.

Protein Expr Purif. 2008 Aug 5;

Authors: Novinec M, Kovačič L, Skrlj N, Turk V, Lenarčič B

Secreted modular calcium-binding (SMOC) proteins are little known members of the BM-40 family of matricellular proteins. SMOC-1 is localized in basement membranes, while SMOC-2 exhibits pro-angiogenic properties and stimulates cell cycle progression via integrin-linked kinase. In this work we have expressed recombinant human SMOCs in inclusion bodies in Escherichia coli. Soluble proteins were prepared by in vitro refolding with a final yield of approximately 3mg of purified SMOCs per liter of bacterial culture. The folding state of the products and their ability to reversibly bind calcium ions were verified by CD spectroscopy. The EF hands of the refolded SMOCs were both functional, one had high affinity for calcium ions (K(d) values in the 0.7-1muM range), while the other had lower affinity (K(d) values 20-25muM). The proteins were also examined for their ability to bind blood serum proteins. Three of the bands specifically retained on SMOC-Sepharose were identified as C-reactive protein, an acute phase protein from the pentraxin family, the basement membrane and elastic fiber-associated fibulin-1, and vitronectin, which is involved in cell adhesion, migration and proliferation and binds numerous extracellular and membrane proteins, including integrins. The interactions were additionally confirmed in solution using purified individual proteins by the method of biotin label transfer from one interacting partner to the other. Their identification is among the first pieces of information about the action of the SMOCs on molecular level and opens new possibilities for future research aimed towards elucidating the physiological roles of these versatile proteins.

PMID: 18708147 [PubMed - as supplied by publisher]

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Impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies of TEM1-beta-lactamase.

Biotechnol J. 2008 Aug 13;

Authors: Margreiter G, Schwanninger M, Bayer K, Obinger C

The enzyme TEM1-beta-lactamase has been used as a model to study the impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies (IBs). The protein has been heterologously expressed in Escherichia coli in fed-batch cultivations at different temperatures (30, 37, and 40 degrees C) as well as induction regimes that guaranteed distinct product formation rates and ratios of soluble to aggregated protein. Additionally, shake flask cultivations at 20, 30, and 37 degrees C were performed. IBs were sampled during the whole bioprocess and structural analysis was performed by attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectroscopy. This work clearly demonstrates that the tested production regimes and rates had no impact on the IB structure, which was characterized by decreased alpha-helical and increased and modified beta-sheet contents compared to the native protein. Moreover, aggregates formed during refolding of IBs by solubilization and simple dilution showed very similar FT-IR spectra suggesting (i) the existence of only one critical folding step from which either aggregation (IB formation) or native folding branches off, and (ii) underlining the important role of the specific amino acid sequence in aggregation. The findings are discussed with respect to the known structure of TEM1-beta-lactamase and the reported kinetics of its (un)folding as well as contradictory data on the effect of cultivation regimes on IB structure(s) of other proteins.

PMID: 18702088 [PubMed - as supplied by publisher]