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• W2015601099 abstract "The explosion in the output of protein structural information over the past ten years has resulted from the convergence of technological breakthroughs in biophysics and molecular biology. Among these advances is the use of recombinant DNA technology to produce large quantities of homogeneous proteins. Indeed, progress in protein overexpression/overproduction technology has had major impacts in other areas aside from structural biology, including the generation of pharmaceuticals and the elucidation of enzymatic catalytic mechanisms. The wide range of eukaryotic cell lines, including yeast, SF9 insect cells, and Chinese hamster ovary (CHO) cells (and their accompanying specialized vectors), that have been developed for heterologous protein overexpression have made it possible to effect production of milligram quantities of complex proteins of great biological interest. Nevertheless, where feasible, the most attractive source of proteins for structural, functional and pharmaceutical applications are still the prokaryotic expression systems (particularly Escherichia coli). No eukaryotic cell line offers the combination of hardiness, rapid growth, ease of genetic manipulation, and simplicity associated with E. coli. Yet, although non-membrane bound prokaryotic protein overexpression in E. coli has become reasonably reliable, results of overexpression of eukaryotic proteins in this organism have traditionally been less predictable. Only part of the problem of eukaryotic protein overproduction in E. coli is the inability of the bacteria to carry out necessary covalent post-translational modifications such as glycosylation or phosphorylation. A more vexing insufficiency is its inability to produce soluble, correctly folded protein. The basis for this deficiency is unclear and probably multifactorial. In some cases, it is believed that the particular eukaryotic protein product is toxic to E. coli growth, creating a selective pressure to prevent the protein's build-up. In others, it is ascribed to the foreign protein's general instability toward proteolysis in the bacterial intracellular environment; however, perhaps the most favoured hypothesis is that failed overexpression results from the target eukaryotic protein's inability to fold efficiently to its stable, functional conformation in E. coli. The net outcome of attempts to overproduce eukaryotic proteins in E. coli is often a very low level of expression and/or formation of significant quantities of insoluble inclusion bodies (aggregates of incorrectly folded protein). Fortunately, several strategies to circumvent the problems of heterologous protein expression in E. coli have been developed, including tightly regulated expression vectors, fusion proteins, and in vitro inclusion body solubilization. Tight regulation of gene expression, exemplified by the use of T7 RNA polymerase and the T7 promoter (found, for example, in the commercially available pET/BL21DE3 system) in principle can reduce the effects of gene toxicity by allowing heterologous expression only in the induction phase [[1]Studier F.W Rosenberg A.H Dunn J.J Dubendorf J.W Use of T7 RNA polymerase to direct expression of cloned genes.Methods Enzymol. 1990; 185 (90340151): 60-89Crossref PubMed Scopus (5901) Google Scholar]. Fusion hybrid protein methods have been particularly powerful. By genetically linking a robust protein such as GST (glutathione-S-transferase) or MBP (maltose binding protein) to the N terminal of the protein of interest, enhanced quantities of soluble protein can be obtained. These fusion products can then be readily purified using glutathione (for GST) or amylose (for MBP) affinity columns. Unfortunately, these hybrid proteins may display non-native behavior (see below). Moreover, although removal of the GST or MBP by proteolytic cleavage may be possible, it is by no means certain and potentially can lead to truncated target proteins [[2]Carrera A.C Alexandrov K Roberts M The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required for anchoring ATP.Proc. Natl. Acad. Sci. USA. 1993; 90 (93133805): 442-446Crossref PubMed Scopus (151) Google Scholar]. Some proteins which are initially isolated as inclusion bodies can be solubilized with detergents or chaotropic salts and then refolded by gradual detergent/salt removal [[3]Noel et al, J.P Tsai M.D Phospholipase A2 engineering. X-ray structural and functional evidence for the interaction of lysine-56 with substrates.Biochemistry. 1991; 30 (92089090): 11801-11811Crossref PubMed Scopus (85) Google Scholar]; however, for most proteins, spontaneous in vitro refolding of denatured proteins to native forms is unsuccessful. Although the above protein expression methods have been dramatically successful in particular cases, they have not been panaceas. Over the past five years, a novel approach, chaperone-assisted protein folding, has been added to the expressionists' arsenal. Chaperone proteins, or chaperonins, were initially characterized as heat shock proteins (hsps), bacterial proteins induced under stress. Several families of chaperonins have now been identified including the hsp90, hsp70, hsp60, proline isomerase, and protein disulfide isomerase families. Most of these families have members throughout the evolutionary tree. Although defining the precise biological roles and mechanisms of action of the chaperonins are very active areas of research, much has already been learned about many of them. A model has been proposed in which DnaK (hsp70 family) and GroEL (hsp60 family) work in succession to effect protein folding in E. coli. That is, the DnaK family functions in early protein maturation and the GroEL family acts at a later stage [[4]Gaitanaris G.A Vysokanov A Hung S.C Gottesman M.E Gragerov A Successive action of Escherichia coli chaperones in vivo.Molecular Microbiology. 1994; 14 (95231293): 861-869Crossref PubMed Scopus (46) Google Scholar]. The hsp60 family, in particular, has been heavily investigated structurally and mechanistically. The first members of this family were identified in the early 1970s as the GroE genes of E. coli and found to participate in bacteriophage λ infection [[5]Georgopoulos C.P Hendrix R.W Casjens S.R Kaiser A.D Host participation in bacteriophage lambda head assembly.J. Mol. Biol. 1973; 76: 45-60Crossref PubMed Scopus (265) Google Scholar]. Subsequently, the GroEL, GroES genes were found to have counterparts in chloroplasts (Rubisco subunit binding protein) [[6]Goloubinoff P Gatenby A.A Lorimer G.H GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli.Nature. 1989; 337 (89082655): 44-47Crossref PubMed Scopus (504) Google Scholar] and mitochondria (hsp60) [[7]Cheng et al, M.Y Horwich A.L Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria.Nature. 1989; 337 (89143719): 620-625Crossref PubMed Scopus (643) Google Scholar]. In eukaryotes, these chaperonins appear to facilitate refolding of target proteins after the latter are transported from the cytosol to the inside of chloroplasts or mitochondria. On the basis of electron microscopy and X-ray crystallography, GroEL has been shown to assemble into a back-to-back dimer of heptamers creating a ‘double-doughnut’ [8Braig et al, K Sigler P.B The crystal structure of the bacterial chaperonin GroEL at 2.8 å.Nature. 1994; 371 (95021709): 578-586Crossref PubMed Scopus (1160) Google Scholar, 9Hendrix R.W Purification and properties of groE, a host protein involved in bacteriophage assembly.J. Mol. Biol. 1979; 129: 375-392Crossref PubMed Scopus (313) Google Scholar]. Proteins in their partially folded form are believed to insert within the doughnut hole where they bind and are prevented from aggregating and forming insoluble inclusion bodies (Figure 1). The release of proteins from GroEL appears to be coupled to GroEL ATPase activity. Furthermore, GroES binding to the complex formed between GroEL and the target protein is also thought to facilitate target protein release, possibly by competitive displacement [[10]Hayer-Hartl M.K Martin J Hartl F.U Asymmetrical interaction of GroEL and GroES in the ATPase cycle of assisted protein folding.Science. 1995; 269 (95365794): 836-841Crossref PubMed Scopus (133) Google Scholar]. Protein-folding intermediates released from GroEL are either thought to continue on to fully folded forms or become re-bound, precluding aggregation. Although elucidation of the mechanistic aspects of the chaperonins, and GroE systems in particular, is ongoing, their utility as protein-folding reagents both in vitro and in vivo is now well demonstrated. As shown in Table 1, they have been used with a variety of monomeric and multimeric proteins to assist protein folding in vitro and expression of soluble protein in vivo. It is this latter aspect which is particularly attractive to investigators encountering difficulty with protein overproduction in E. coli. By boosting the levels of chaperonins from 1% (the approximate endogenous level) to greater than 10% total E. coli cell protein, significantly enhanced soluble protein expression has been observed. Both the hsp60 and hsp70 families have been used successfully to increase soluble target protein expression (Table 1). As shown, a wide variety of enzymes, peptide hormones, and cytokines have been produced in this manner. Table 1 also shows the approximate expression enhancements and an indication of whether the expressed protein has been purified and/or characterized. The general technique for carrying out chaperonin-assisted protein expression involves transforming E. coli with separate chaperone-expressing and target-protein-expressing plasmids bearing distinct antibiotic resistance functions and replication origins (for an example, see Figure 2). In this way, both plasmids may be stably maintained in the cell. Typically, promoters inducible with IPTG have been used to drive gene expression of both target proteins and chaperonins so that they are induced concomitantly.Table 1Examples of chaperonin-assisted protein expression.Target proteinChaperone familyIn vitro/In vivoSolublechap/ Solublectrl∗Purified/ Characterized†ReferencesCskGroE‡In vivo4–8Both12Caspers P Stieger M Burn P Overproduction of bacterial chaperones improves the solubility of recombinant protein tyrosine kinases in E. coli.Cell. Mol. Biol. 1994; 40 (95072497): 635-644PubMed Google Scholar, 13Amrein et al, K.E Burn P Purification and characterization of human recombinant p50csk protein tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.Proc. Natl. Acad. Sci. USA. 1995; 92 (95166765): 1048-1052Crossref PubMed Scopus (148) Google Scholar, 14Cole P.A Burn P Takacs B Walsh C.T Evaluation of the catalytic mechanism of recombinant human Csk (C-terminal Src kinase) using nucleotide analogs and viscosity effects.J. Biol. Chem. 1994; 269 (95074117): 30880-30887Abstract Full Text PDF PubMed Google ScholarLckGroE/DnaK#In vivoN.R.§N.R.[12]Caspers P Stieger M Burn P Overproduction of bacterial chaperones improves the solubility of recombinant protein tyrosine kinases in E. coli.Cell. Mol. Biol. 1994; 40 (95072497): 635-644PubMed Google ScholarFynGroE/DnaKIn vivoN.R.N.R.[12]Caspers P Stieger M Burn P Overproduction of bacterial chaperones improves the solubility of recombinant protein tyrosine kinases in E. coli.Cell. Mol. Biol. 1994; 40 (95072497): 635-644PubMed Google ScholarDihydrofolate reductaseGroEBoth3–4Both[17]Dale G.E Schonfeld H.-J Langen H Stieger M Increased solubility of trimethoprim-resistant type S1 DHFR from Staphylococcus aureus in Escherichia coli cells overproducing the chaperonins GroEL and GroES.Protein Eng. 1994; 7 (95062140): 925-931Crossref PubMed Scopus (64) Google Scholarα-Ketoacid∗∗ dehydrogenase kinaseGroEIn vivo3–4Both[18]Davie et al, J.R Lau K.S Expression and characterization of branched-chain α-ketoacid dehydrogenase kinase from the rat.J. Biol. Chem. 1995; 270 (95378233): 19861-19867Crossref PubMed Scopus (49) Google ScholarCitrate SynthaseGroEIn vitro––[19]Zhi W Landry S.J Gierasch L.M Srere P.A Renaturation of citrate synthase: influence of denaturant and folding assistants.Protein Sci. 1992; 1 (93278281): 522-529Crossref PubMed Scopus (91) Google ScholarLactate dehydrogenaseGroEIn vitro––[20]Badcoe et al, I.G Clarke A.R Binding of a chaperonin to the folding intermediates of lactate dehydrogenase.Biochemistry. 1991; 30 (91369931): 9195-9200Crossref PubMed Scopus (138) Google ScholarTryptophanaseGroEIn vitro––[21]Mizobata T Akiyama Y Ito K Yumoto N Kawata Y Effects of the Chaperonin GroE on the refolding of tryptophanase from Escherichia coli.J. Biol. Chem. 1992; 267 (92388132): 17773-17779Abstract Full Text PDF PubMed Google ScholarOrnithine transcarbamylaseGroEBothN.R.N.R.[22]Zheng X Rosenberg L.E Kalousek F Fenton W.A GroEL, GroES, and ATP-dependent folding and spontaneous assembly of ornithine transcarbamylase.J. Biol. Chem. 1993; 268 (93216697): 7489-7493Abstract Full Text PDF PubMed Google ScholarPre-β-lactamaseGroEIn vitro––[23]Laminet A.A Ziegelhoffer T Georgopoulos C Pluckthun A The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the β-lactamase precursor.EMBO J. 1990; 9 (90291998): 2315-2319Crossref PubMed Scopus (196) Google ScholarRhodanaseGroEIn vitro––[24]Mendoza J.A Rogers E Lorimer G.H Horowitz P.M Chaperonins facilitate the in vitro folding of monomeric rhodanese.J. Biol. Chem. 1991; 266 (91302323): 13044-13049Abstract Full Text PDF PubMed Google ScholarRubiscoGroEBothN.R.Characterized[25]Goloubinoff P Christeller J.T Gatenby A.A Lorimer G.H Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP.Nature. 1989; 342: 884-889Crossref PubMed Scopus (530) Google Scholarα-Ketoacid†† decarboxylaseGroEIn vivoN.R.Both[26]Wynn R.M Davie J.R Cox R.P Chuang D.T Chaperonins GroEL and GroES promote assembly of heterotetramers (α2β2) of mammalian mitochondrial branched-chain α-keto acid decarboxylase in Escherichia coli.J. Biol. Chem. 1992; 267 (92316908): 12400-12403Abstract Full Text PDF PubMed Google ScholarNO synthaseGroEIn vivo>10Both[27]Roman L.J Sheta E.A Martasek P Gross S.S Liu Q Masters B.S High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli.Proc. Natl. Acad. Sci, USA. 1995; 92 (95396809): 8428-8432Crossref PubMed Scopus (243) Google ScholarGranulocyte colony stimulating factorDnaKIn vivo6–8N.R.[28]Perez-Perez J Martinez-Caja C Barbero J.L Gutierrez J DnaK/DnaJ supplementation improves the periplasmic production of human granulocyte-colon stimulating factor in Escherichia coli.Biochem. Biophys. Res. Commun. 1995; 210 (95275306): 524-529Crossref PubMed Scopus (46) Google ScholarHuman growth hormoneDnakIn vivo2–8N.R.[29]Blum P Velligan M Lin N Matin A DnaK-mediated alterations in human growth hormone protein inclusion bodies.Biotechnology. 1992; 10: 301-304Crossref PubMed Scopus (57) Google Scholar∗Relative amount of soluble target protein expressed in the presence of chaperonin compared with no chaperone, estimated in the references or by this author, only for the in vivo expression systems. †Indication of whether proteins expressed in vivo were either purified to near homogeneity and/or characterized by enzyme assay and shown to have normal behavior. ‡Includes co-expression of GroES and GroEL. §Not reported. #May include co-expression of DnaJ and GrpE. ∗∗Mammalian mitochondrial branched-chain α-ketoacid dehydrogenase kinase. ††Mammalian mitochondrial branched-chain α-ketoacid decarboxylase. Open table in a new tab ∗Relative amount of soluble target protein expressed in the presence of chaperonin compared with no chaperone, estimated in the references or by this author, only for the in vivo expression systems. †Indication of whether proteins expressed in vivo were either purified to near homogeneity and/or characterized by enzyme assay and shown to have normal behavior. ‡Includes co-expression of GroES and GroEL. §Not reported. #May include co-expression of DnaJ and GrpE. ∗∗Mammalian mitochondrial branched-chain α-ketoacid dehydrogenase kinase. ††Mammalian mitochondrial branched-chain α-ketoacid decarboxylase. An application of the GroE methodology in our mechanistic studies on the protein tyrosine kinase Csk will serve as an example, illustrating both its power as well as some potential pitfalls. We were interested in studying the detailed enzymatic mechanism of Csk and required milligram quantities of pure protein. E. coli was especially desirable as the source of the recombinant enzyme not only for the traditional reasons described above but because endogenous protein tyrosine kinases are thought to be absent from this organism. This is particularly important in characterizing catalytically impaired Csk mutants where contaminating background kinase activities, despite purification efforts, can interfere with mechanistic interpretations (see below). Overproduction of a GST–Csk recombinant fusion protein from E. coli had been reported but this hybrid protein was shown to be catalytically defective compared with wild type (with specific activity reportedly reduced by 16-fold) [[11]Bougeret C Rothhut B Pascale J Fischer S Benarous R Recombinant Csk expressed in Escherichia coli is autophosphorylated on tyrosine residue(s).Oncogene. 1993; 8 (93241724): 1241-1247PubMed Google Scholar]. Furthermore, bacterial expression of standard full-length Csk led to very low proportions of soluble protein (ca. 10–20%) [12Caspers P Stieger M Burn P Overproduction of bacterial chaperones improves the solubility of recombinant protein tyrosine kinases in E. coli.Cell. Mol. Biol. 1994; 40 (95072497): 635-644PubMed Google Scholar, 13Amrein et al, K.E Burn P Purification and characterization of human recombinant p50csk protein tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.Proc. Natl. Acad. Sci. USA. 1995; 92 (95166765): 1048-1052Crossref PubMed Scopus (148) Google Scholar]. In contrast, when the GroESL plasmid (pREP4–groESL) was co-introduced with the Csk expressing plasmid (pDS56/RBSII–csk) into E. coli (see Figure 2), the majority of the expressed Csk protein (ca. 70–80%) was found to be soluble under standard growth conditions (induction with 1 mM IPTG at A600=0.6–0.9, 37°C, 4 h) [[13]Amrein et al, K.E Burn P Purification and characterization of human recombinant p50csk protein tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.Proc. Natl. Acad. Sci. USA. 1995; 92 (95166765): 1048-1052Crossref PubMed Scopus (148) Google Scholar]. A Coomassie stained SDS PAGE gel of the crude cell lysate shows that approximately a 1:1 ratio (w/w) of wild-type Csk to GroEL protein is produced, each representing ca. 20% of total cell protein [[13]Amrein et al, K.E Burn P Purification and characterization of human recombinant p50csk protein tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.Proc. Natl. Acad. Sci. USA. 1995; 92 (95166765): 1048-1052Crossref PubMed Scopus (148) Google Scholar]; however, the molar quantities of Csk and GroEL are not the same (as in a 1:1 complex) as GroEL functions as a double heptamer. This encouraging result was tempered by the fact that in our hands, using the published purification of recombinant Csk from E. coli cell extracts [[13]Amrein et al, K.E Burn P Purification and characterization of human recombinant p50csk protein tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.Proc. Natl. Acad. Sci. USA. 1995; 92 (95166765): 1048-1052Crossref PubMed Scopus (148) Google Scholar], much of the Csk (>50%) was lost in the first chromatographic step (Phospho-Ultrogel), appearing in the elution void volume along with GroEL. The basis for this Csk loss was initially unclear; that is, it was not known if the fraction of Csk protein that did not bind to the Phospho-Ultrogel was non-native like. Nevertheless, the Csk protein which did adhere to the Phospho-Ultrogel resin could be eluted with a salt gradient to produce a peak of quite pure protein, nearly free from GroEL contamination. This purified recombinant Csk had similar kinetic parameters to those reported for endogenous wild type Csk, suggesting that the purified recombinant enzyme was well-behaved [[14]Cole P.A Burn P Takacs B Walsh C.T Evaluation of the catalytic mechanism of recombinant human Csk (C-terminal Src kinase) using nucleotide analogs and viscosity effects.J. Biol. Chem. 1994; 269 (95074117): 30880-30887Abstract Full Text PDF PubMed Google Scholar]. In an effort to simplify the published Csk purification procedure, as well as to improve recovery, we made use of a phosphotyrosine affinity column in the initial chromatographic step [[15]Koegel M Kypta R.M Bergman M Alitalo K Courtneidge Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85.Biochem. J. 1994; 302 (95031912): 737-744PubMed Google Scholar]. In contrast to Phospho-Ultrogel, nearly all (>90%) of the Csk protein stuck to the phosphotyrosine column whereas the GroEL was removed in the void. Csk could be eluted readily with a salt gradient in a sharp peak. This resulted in essentially homogeneous Csk in quantities of 25 mg L−1 E. coli cell culture (7 mg per g cell paste, about a threefold higher recovery then reported in [[13]Amrein et al, K.E Burn P Purification and characterization of human recombinant p50csk protein tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.Proc. Natl. Acad. Sci. USA. 1995; 92 (95166765): 1048-1052Crossref PubMed Scopus (148) Google Scholar]). Moreover, Csk recovered from this purification was found to behave identically, in mechanistic studies, to material obtained from the original purification scheme. Therefore, it seemed likely that losses suffered in the Phospho-Ultrogel column purification step were due to a partitioning in Csk binding between GroEL and Phospho-Ultrogel. In contrast, with the high affinity phosphotyrosine-Csk SH2 interaction, Csk column binding is favored, allowing complete separation from GroEL. We have also used the same expression system to produce a Csk mutant, D314E [[16]Cole P.A Grace M.R Phillips R.S Burn P Walsh C.T The role of the catalytic base in the protein tyrosine kinase Csk.J. Biol. Chem. 1995; 270 (95403393): 22105-22108Crossref PubMed Scopus (56) Google Scholar]. Csk D314E was prepared to assess the role of the proposed catalytic base, Asp314, in the reaction mechanism. The mutant enzyme had a kcat which was reduced by approximately 10 000-fold compared with wild-type Csk [[16]Cole P.A Grace M.R Phillips R.S Burn P Walsh C.T The role of the catalytic base in the protein tyrosine kinase Csk.J. Biol. Chem. 1995; 270 (95403393): 22105-22108Crossref PubMed Scopus (56) Google Scholar]. Nevertheless, we were able to accurately measure the mutant kinetic parameters of D314E Csk without complications of background tyrosine kinase activity because of the lack of endogenous tyrosine kinases in E. coli. These studies allowed us to propose a new functional role for Asp314, as activator of the γ-phosphate of ATP [[16]Cole P.A Grace M.R Phillips R.S Burn P Walsh C.T The role of the catalytic base in the protein tyrosine kinase Csk.J. Biol. Chem. 1995; 270 (95403393): 22105-22108Crossref PubMed Scopus (56) Google Scholar]. Although the DNA constructs for Csk D314A and D314N were also prepared, they failed to afford significant protein expression (soluble or insoluble) under these GroE conditions. One wonders if the application of concomitant chaperonin expression may make the conditions for poorly expressed proteins even worse because of competition in vivo for the synthetic machinery. In principle, such competition might be alleviated in the future if constructs for the chaperonins could be titrated and induced prior to the target proteins by using different, regulatable promoters. Conclusions: Overproduction of recombinant proteins in E. coli continues to be an important step in their biochemical and structural characterization. Along with other methods, chaperonins contribute significantly to improvements in protein over-production. Nevertheless, technical difficulties still occur with the use of chaperonins, both in expression and purification of the target proteins. As insights into the molecular mechanisms of chaperonin function are learned, and greater biochemical experience with their applications accrues, general principles regarding chaperonin activities will be defined. The formulation of such principles should make chaperonin-assisted protein expression an even more powerful method. I thank the Howard Hughes Medical Institute for a post-doctoral fellowship. I thank Prof Chris Walsh and members of the Walsh group for helpful suggestions. Philip A Cole, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA." @default.
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