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1.
Family of G protein {alpha} chains: amphipathic analysis and predicted structure of functional domains 总被引:7,自引:0,他引:7
Masters Susan B.; Stroud Robert M.; Bourne Henry R. 《Protein engineering, design & selection : PEDS》1986,1(1):47-54
The G proteins transduce hormonal and other signals into regulationof enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase.Each G protein contains an subunit that binds and hydrolyzesguanine nucleotides and interacts with ß subunitsand specific receptor and effector proteins. Amphipathic andsecondary structure analysis of the primary sequences of fivedifferent chains (bovine s, t1 and t2, mouse i, and rat o)predicted the secondary structure of a composite chain (avg).The chains contain four short regions of sequence homologousto regions in the GDP binding domain of bacterial elongationfactor Tu (EF-Tu). Similarities between the predicted secondarystructures of these regions in avg and the known secondary structureof EF-Tu allowed us to construct a three-dimensional model ofthe GDP binding domain of avg. Identification of the GDP bindingdomain of avg defined three additional domains in the compositepolypeptide. The first includes the amino terminal 41 residuesof avg, with a predicted am phipathic helical structure; thisdomain may control binding of the chains to the ßcomplex. The second domain, containing predicted ßstrands and helices, several of which are strongly amphipathic,probably contains sequences responsible for interaction of chains with effector enzymes. The predicted structure of thethird domain, containing the carhoxy terminal 100 amino acids,is predominantly ß sheet with an amphipathic helixat the carboxy terminus. We propose that this domain is reponsiblefor receptor binding. Our model should help direct further experimentsinto the structure and function of the G protein chain. 相似文献
2.
Srinivasulu Sonati; Malavalli Ashok; Prabhakaran Muthuchidambaran; Nagel Ronald L.; Acharya A.Seetharama 《Protein engineering, design & selection : PEDS》1999,12(12):1105-1111
Mouse 130-horse 31141 chimeric -chain, a semisyntheticsuper-inhibitory -chain, inhibits ßS-chain dependent polymerizationbetter than both parent -chains. Although contact site sequencedifferences are absent in the 130 region of the chimericchain, the four sequence differences of the region 17-22 couldinduce perturbations of the side chains at 16, 20 and 23, thethree contact sites of the region. A synergistic complementationof such contact site perturbation with that of horse 31141probably results in the super-inhibitory activity of the chimeric-chain. The inhibitory contact site sequence differences, bythemselves, could also exhibit similar synergistic complementation.Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin(LM) mutation [His20()Gln], a contact site sequence difference,engineered into humanhorse chimeric -chain has been investigatedto map such a synergistic complementation. Gln20() has littleeffect on the O2 affinity of HbS, but in humanhorse chimeric-chain it reduces the O2 affinity slightly. In the chimeric-chain, Gln20() increased sensitivity of the ßßcleft for the DPG influence, reflecting a cross-talk betweenthe 1ß1 interface and ßß cleft in this semisyntheticchimeric HbS. In the human -chain frame, the polymerizationinhibitory activity of Gln20() is higher compared with horse130, but lower than mouse 130. Gln20() synergisticallycomplements the inhibitory propensity of horse 31141.However, the inhibitory activity of LMhorse chimeric-chain is still lower than that of mousehorse chimeric-chain. Therefore, perturbation of multiple contact sites inthe 130 region of the mousehorse chimeric -chainand its linkage with the inhibitory propensity of horse 31141has been now invoked to explain the super-inhibitory activityof the chimeric -chain. The `linkage-map' of contact sites canserve as a blueprint for designing synergistic complementationof multiple contact sites into -chains as a strategy for generatingsuper-inhibitory antisickling hemoglobins for gene therapy ofsickle cell disease. 相似文献
3.
Jallat S.; Carvallo D.; Tessier L.H.; Roecklin D.; Roitsch C.; Ogushi F.; Crystal R.G.; Courtney M. 《Protein engineering, design & selection : PEDS》1986,1(1):29-35
Seven active site variants of human 1-antitrypsin (1AT) wereproduced in Escherichia coli following site-specific mutagenesisof the 1AT complementary DNA. 1AT (Ala 358), 1AT (Ile358 and1AT (Val358), were efficient inhibitors of both neutrophil andpancreatic elastases, but not of cathepsin G. 1AT (Ala358, Val358)and 1AT (Phe358 specifically inhibited pancreatic elastase andcathepsin G respectively. The most potent inhibitor of neutrophilelastase was 1AT (Leu358), which also proved to be effectiveagainst cathepsin G. The 1AT (Arg358) variant inactivated thrombinwith kinetics similar to antithrombin III in the presence ofheparin. Electrophoretic analysis showed that SDS-stable highmol. wt complexes were formed between the mutant inhibitorsand the cognate proteases in each case. These data indicatethat effective inhibition occurs when the 1AT P1 residue (position358) corresponds to the primary specificity of the target protease.Moreover, alteration of the P3 residue (position 356) can furthermodify the reactivity of the inhibitor. Two of the variantshave therapeutic potential: 1AT (Leu358 may be more useful thanplasma 1AT in the treatment of destructive lung disorders and1 (Arg358 could be effective in the control of thrombosis. 相似文献
4.
Production and characterization of anti-human interferon {gamma} receptor antibody fragments that inhibit cytokine binding to the receptor 总被引:1,自引:0,他引:1
Bridges Angela; Stuart Fiona; Spth Julia; Lang Stefan; Henke Christoph; Birch Ashley; Robinson John A. 《Protein engineering, design & selection : PEDS》1996,9(4):365-370
Three single-chain antibody fragments that recognize the extracellularhuman interferon receptor -chain (IFNR), and inhibit the bindingof human IFN, have been produced in Escherichia coli. Thesefragments are derived from murine anti-receptor monoclonal antibodies,and comprise the variable heavy (VH) domain linked to the variablelight (VL) chain through a 15 amino acid linker [(GGGGS)3].Using surface plasmon resonance technology (BIAcore), the solubleproteins were shown to retain a high affinity for recombinantIFNR, and by radioimmunoassay to possess high inhibitory activitytowards IFN-binding to human Raji cells. The antibody fragmentsmost likely recognize epitopes that overlap the cytokine bindingsite on the receptor surface. Attempts to dissect further theantibodies to isolated VH- and VL-chains and to synthetic linearand cyclic peptides derived from the individual complementaritydetermining regions failed to afford fragments with significantIFNR binding affinity. Nevertheless, these native-like variableregion fragments and petidomimetics derived from them are ofinterest in the design of novel IFNR antagonists. 相似文献
5.
Bhat Suraj P.; Nandy Partha; Srinivasan Anu; Cheng David; Sitay Anthony 《Protein engineering, design & selection : PEDS》1996,9(8):713-718
A-Crystallin and Ains-crystallin are derived from the A-crystallingene via alternative splicing. They are identical except forthe presence of a polypeptide, 23 amino acids long, encodedby the insert exon. Evolutionary logic would suggestthat the insertion of a 23 amino acid peptide in the middleof A-crystallin, a protein evolving more slowly than eitherhistone H1, cytochrome c or hemoglobin, would lead to appreciablestructural and functional changes. However, based on physico-chemicalstudies, it is presently believed that A-crystallin and Ains-crystallinare functionally equivalent and that the presence of the insertpeptide in AIns-crystallin is inconsequential. We report herethat the independent expression of recombinant AIns-crystallin,and not A-crystallin, inhibits growth of the bacterial host.These observations were confirmed in co-expression experiments,wherein both the proteins were expressed in the same cell. Interestingly,growth inhibition is reversible. Importantly, the data demonstratethat it is catalytic amounts and not the gross accumulationof AIns-crystalline which causes growth inhibition. Given theprior knowledge that A-crystallin and AIns-crystallin differby a peptide of 23 amino acids, these data suggest that theinsert peptide in AIns-crystallin imparts propertieson this protein that are different from A-crystallin. 相似文献
6.
Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase 总被引:1,自引:0,他引:1
Libby Carol Baker; Cornett Catherine A.G.; Reilly Peter J.; Ford Clark 《Protein engineering, design & selection : PEDS》1994,7(9):1109-1114
Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1471 and 509616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441471) in the crystal structurewraps around an /-barrel formed by residues 1440. Thesecond (residues 472508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466512 (GA1), 485512 (GA2) and 466483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities. 相似文献
7.
Luger Karolin; Szadkowski Halina; Kirschner Kasper 《Protein engineering, design & selection : PEDS》1990,3(4):249-258
Protein sequences containing redundant segments of secondarystructure at both termini have the choice a priori of foldinginto several possible circularly permuted variants of the wild-typetertiary structure. To test this hypothesis the gene of phosphoribosylanthranilate isomerase from yeast, which is a single-domain8-fold ß barrel protein, was modified to produce a10-fold ß homologue in Escherichia coli. It containeda duplicate of the two C-terminal ß units of supersecondarystructure fused to its N-terminus. Most of the protein was recoveredfrom the insoluble fraction of disrupted cells by dissolutionin guanidinium chloride solutions and refolding. Pristine proteinwas purified from the soluble fraction. The purified (ß)10proteins were enzymically almost fully active. Absorbance, fluorescenceand circular dichroism spectra as well as the reversible unfoldingbehaviour of both proteins were also very similar to the propertiesof the original (ß)8 protein. Digestion with endopeptidasesconverted both the pristine and the refolded (ß)10variant to the same large fragment that had the N-terminal sequenceand mol. wt of the wild-type ß)8 protein. The datasuggest that the folding of the (ß)10 variant is controlledthermodynamically both in vivo and in vitro. 相似文献
8.
Improvement of turn structure prediction by molecular dynamics: a case study of {alpha}1-purothionin
Because of the problems in predicting a correct conformationfor loop regions in homology-based prediction, disagreementsare often found between the predicted models and the refinedX-ray structures of the same protein in loop regions. Such asituation has been encountered for 1-purothionin (1-PT). Hence,attempts have been made to improve the predicted model of 1PTby limited molecular dynamics using both AMBER and XPLOR. Withmolecular dynamics, the previously predicted incorrect turnregion reverts to the correct conformation as seen in the X-rayrefined structure. In contrast to the model which is not subjectedto molecular dynamics, the improved model refines with the X-raydata of 1PT in fewer cycles, without any manual rebuilding andwith comparable or better refinement statistics. Also, the improvedmodel serves as a better starting model in the determinationof the structure with the molecular replacement methods. 相似文献
9.
To understand the functional roles of Cys residues in the subunitof tryptophan synthase from Escherichia coli, single mutantsof the subunit, in which each of the three Cys residues wassubstituted with Ser, Gly, Ala or Val, were constructed by site-directedmutagenesis. The effects of the substitutions on the functionof tryptophan synthase were investigated by activity measurements,calorimetric measurements of association with the ßsubunit and steadystate kinetic analysis of catalysis. Althoughthe three Cys residues are located away from the apparentlyimportant parts for enzymatic activity, substitutions at position81 by Ser, Ala or Val caused decreases in the intrinsic activityof the subunit. Furthermore, Cys81Ser and Cys81Val reducedstimulation activities in the and ß reactions dueto formation of a complex with the ß subunit. Thelower stimulation activities of the mutant proteins were notcorrelated with their abilities to associate with the ßsubunit but were correlated with decreases in kcat. The presentresults suggest that position 81 plays an indirectly importantrole in the activity of the subunit itself and the mutual activationmechanism of the complex. 相似文献
10.
Twelve different (/ß)8-barrel enzymes belonging tothree structurally distinct families were found to contain,near the C-terminus of their strand ß5, a conservedinvariant glutamic acid residue that plays an important functionalrole in each of these enzymes. The search was based on the ideathat a conserved sequence region of an (/ß)8-barrelenzyme should be more or less conserved also in the equivalentpart of the structure of the other enzymes with this foldingmotif owing to their mutual evolutionary relatedness. For thispurpose, the sequence region around the well conserved fifthß-strand of a-amylase containing catalytic glutamate(Glu230, Aspergillus oryzae -amylase numbering), was used asthe sequence-structural template. The isolated sequence stretchesof the 12 (/ß)8-barrels are discussed from both thesequence-structural and the evolutionary point of view, theinvariant glutamate residue being proposed to be a joining featureof the studied group of enzymes remaining from their ancestral(/ß)8-barrel 相似文献
11.
Manjula Belur N.; Malavalli Ashok; Prabhakaran Muthuchidambaram; Friedman Joel M.; Acharya A.Seetharama 《Protein engineering, design & selection : PEDS》2001,14(5):359-366
The Asn108ßLys mutation in hemoglobin (HbPresbyterianmutation) endows a low O2 affinity-inducing propensity to theprotein. Introduction of a fumaryl cross-bridge between itstwo 99 lysine residues also induces a low O2 affinity into HbA.We have now engineered an -fumaryl cross-bridge into Hb-Presbyterianto determine the synergy or additivity, if any, that can beachieved between these two low O2 affinity-inducing structuralperturbations. Despite the presence of the additional -aminogroup of Lys108(ß) within the central cavity, the-amino group of Lys99() of deoxy Hb-Presbyterian retained highselectivity for -fumaryl cross-bridging, with an overall efficiencycomparable to that with HbA. The -fumaryl cross-linking of Hb-Presbyterianreduced its O2 affinity much more significantly than that observedwith HbA, indicating a synergy between the two low O2 affinity-inducingstructural perturbations. Apparently, the -fumaryl cross-bridgein Hb-Presbyterian activates part of the latent low O2 affinity-inducingpotential of Lys108(ß) that is generally activatedin the presence of chloride. The synergy between the Asn108(ß)Lysmutation and the -fumaryl cross-bridging was conserved in thepresence of chloride, but not in the presence of DPG. Furthermore,in the presence of chloride and DPG, -fumaryl Hb-Presbyterianaccessed a low O2 affinity T-state that is accessed by HbA,-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectricfocusing analysis suggested that the -fumaryl cross-linkingof Hb-Presbyterian induces changes in the ionization behaviorof one or more of the functional groups neighboring Lys99()and Lys108(ß) [presumably His103() and/or Glu101(ß)]to compensate for the extra positive charge of Lys108(ß).Molecular modeling studies identified two potential chloridebinding sites per ß dimer within the middle of thecentral cavity of -fumaryl HbA involving residues His103(),Arg104(ß) and Asn108(ß). The affinity ofthese sites is increased in -fumaryl Hb-Presbyterian as a resultof the Asn108(ß)Lys mutation. Thus, the results ofthe present study suggest that the enhanced neutralization ofthe positive charges in the middle of the central cavity ofHb achieved by these two electrostatic modifications, one (the-fumaryl cross-bridge) acting directly and the other (the Presbyterianmutation) acting indirectly through the mediation of chlorideion binding, facilitates the - fumaryl-Hb Presbyterian to accessa low O2 affinity T-state structure much more readily than eitherHb-Presbyterian or -fumaryl HbA. 相似文献
12.
Henrick K.; Blow D.M.; Carrell H.L.; Glusker J.P. 《Protein engineering, design & selection : PEDS》1987,1(6):467-469
The C backbones of the glucose isomerase molecules of Streptomycesrubiginosus and Arthrobacter have been determined by X-ray crystallographyand compared. Each molecule is a tetramer of eight-stranded/ß barrels, and the mode of association of the tetramersis identical in each case. The Arthrobacter electron densityshows four additional amino acids at the carboxyl terminus.There is also an insertion of six amino acids at position 277,and two individual insertions at about positions 348 and 357(numbering according to the Streptomyces structure). There isa close structural homology throughout the whole molecule, whichis most accurate up to position 325. The r.m.s. displacementfor 315 homologous C positions up to this position is 0.92 Å. 相似文献
13.
A Pore-forming protein with a protease-activated trigger 总被引:3,自引:0,他引:3
Hemolysin (HL) is a 293 amino acid pore-forming toxin, whichis secreted as a water-soluble monomer by Staphylococcus aureus.By forming a hexameric pore, HL damages the plasma membranesof target cells. Previous studies established that HL proteinswith nicks near the midpoint of a central glycine-rich loopare held together by a domain-domain interaction and are hemolyticallyactive. In contrast, HL proteins comprising two HL truncationmutants that overlap in the central loop have no or greatlyreduced pore-forming activity, even though the two chains againform a tight complex. Based on these findings, overlap mutantshave now been designed that are activated when redundant aminoacids in the loop are removed by proteases. Further, the identityof the activating enzyme can be specified by additional mutagenesisof the protease recognition site in the overlap sequence. Mutantsof aHL that are activated by tumor-associated proteases mightbe useful components of immunotoxins 相似文献
14.
The prediction of the side-chain positions of proteins of knowntertiary backbone structure was accomplished by a combinationof neural networks and a simulated annealing method. Neuralnetworks were used to generate distributions of side-chain dihedralangles. By eliminating network outputs with low activities,we were able to generate a reduced conformational space in whichMonte Carlosimulated annealing was carried out to optimize side-chainpositions. In this study of 12 proteins, the average fractionsof correct 1 2 a nd combined 1 and 2 (to within 40° of actualstructure) were 82, 72 and 68% respectively. 相似文献
15.
Sierks Michael R.; Ford Clark; Reilly Peter J.; Svensson Birte 《Protein engineering, design & selection : PEDS》1993,6(1):75-79
Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177 His, Trp178 Arg and Asn182 Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in kcat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. KM valuesobtained with the Trp178 Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change kcat or KM values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex. 相似文献
16.
Semi-empirical simulation of Zn/Cd binding site preference in the metal binding domains of mammalian metallothionein 总被引:1,自引:0,他引:1
Metallothionein, a two-domain protein, naturally binds sevengram atoms of divalent ions such as Zn and Cd. Four of the metals(Ml, M5, M6 and M7) are found in the -domain and three (M2,M3 and M4) in the ß-domain. Previous studies haveshown that metals in the -domain are more readily exchangeable,and the level of avidity is site specific. By semi-empiricalMNDO modified neglect of diatomic overlap calculations, we foundthe tendency of binding energy for Cd to be M3 > M2 >M4 in the ß-cluster and M5 > M7 > Ml, M6 inthe -cluster. Thus, the replacement of Zn by Cd can be expectedto follow the order M4 M2 M3 in the ß-domain andMS M7 M1 or M6 in the -domain. This is reflected by energydifferences computed with a series of simulated structures derivedfrom either X-ray crystallography or NMR coordinates. 相似文献
17.
Human tumour necrosis factors (hTNFs) and ß are relatedpleiotropic cytokines which share many activities and competewith each other for binding to two receptor components on manycell types. Although structural and biological data indicatethat the active form of hTNF- may be a symmetrical trimer, themanner in which hTNFs interact with their receptors to triggera myriad of cell type-dependent responses is not clear. A combinationof chemical modification, epitope mapping and site-directedmutagenesis approaches suggest that at least four distinct peptidesequences are Important for the biological activity of hTNF-.In particular, certain peptide sequences between amino acidpositions 11 and 35 in hTNF- appear to be critical for receptorbinding and triggering biological responses. The recent cloningof the two hTNF-/ß receptors opens the way for precisemapping of the functional domains in hTNFs 相似文献
18.
Srinivasan N.; Antonelli Marcelo; Jacob Germaine; Korn Iris; Romero Francisco; Jedlicki Ana; Dhanaraj V.; Sayed Muhammed F.-R.; Blundell Tom L.; Allende Catherine C.; Allende Jorge E. 《Protein engineering, design & selection : PEDS》1999,12(2):119-127
The catalytic subunit of protein kinase casein kinase 2 (CK2),which has specificity for both ATP and GTP, shows significantamino acid sequence similarity to the cyclin-dependent kinase2 (CDK2). We constructed site-directed mutants of CK2 and useda three-dimensional model to investigate the basis for the dualspecificity. Introduction of Phe and Gly at positions 50 and51, in order to restore the pattern of the glycine-rich motif,did not seriously affect the specificity for ATP or GTP. Weshow that the dual specificity probably originates from theloop situated around the position His115 to Asp120 (HVNNTD).The insertion of a residue in this loop in CK2 subunits, comparedwith CDK2 and other kinases, might orient the backbone to interactwith the base A and G; this insertion is conserved in all knownCK2. The mutant N118, the design of which was based on the modelling,showed reduced affinity for GTP as predicted from the model.Other mutants were intended to probe the integrity of the catalyticloop, alter the polarity of a buried residue and explore theimportance of the carboxy terminus. Introduction of Arg to replaceAsn189, which is mapped on the activation loop, results in amutant with decreased kcat, possibly as a result of disruptionof the interaction between this residue and basic residues inthe vicinity. Truncation at position 331 eliminates the last60 residues of the subunit and this mutant has a reduced catalyticefficiency compared with the wild-type. Catalytic efficiencyis restored in the truncation mutant by the replacement of apotentially buried Glu at position 252 by Lys, probably owingto a higher stability resulting from the formation of a saltbridge between Lys252 and Asp208. 相似文献
19.
Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha 总被引:1,自引:0,他引:1
Yamagishi Jun-ichi; Kawashima Hitoshi; Matsuo Noriyuki; Ohue Mayumi; Yamayoshi Michiko; Fukui Toshikazu; Kotani Hirotada; Furuta Ryuji; Nakano Katsuji; Yamada Masaaki 《Protein engineering, design & selection : PEDS》1990,3(8):713-719
To determine the region of human tumor necrosis factor-alpha(TNF-), essential for cytotoxic activity against mouse L-M cells,single amino-acid-substituted TNF- mutant proteins (muteins)were produced in Escherichia coli by protein engineering techniques.An expression plasmid for TNF- was mutagenized by passage throughan E.coli mutD5 mutator strain and by oligonucleotide-directedmutagenesis. Approximately 100 single amino-acid-substitutedTNF- muteins were produced and assayed for cytotoxic activity.The cytotoxic activities of purified TNF- muteins, e.g. TNF-31T,-32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were < 1%of that of parent TNF-. These results indicate that the integrityof at least four distinct regions of the TNF- molecule is requiredfor full biological activity. These regions are designated asfollows: region I, from position 30 to 32; region II, from position82 to 89; region III, from position 115 to 117; region FV, fromposition 141 to 146. In addition, TNF-141Y could not completelycompete with parent TNF- for binding to the receptor. This demonstratesthat region IV, and at least aspartk acid at position 141, mustbe involved in the TNF receptor binding site. 相似文献
20.
Pan Aihua; Tie Feng; Yang Meizhu; Luo Jingchu; Wang Zhengxing; Ding Xiang; Li Lingyuan; Chen Zhangliang; Ru Binggen 《Protein engineering, design & selection : PEDS》1993,6(7):755-762
Metallothioneins (MT) are low molecular weight, cysteinerich,metal-binding proteins. An MT molecule contains two domainswhich appear to act independentlyan -domain, which ischaracterized by cadmium-binding, and a ß-domain,which binds preferentially to copper. Based on this conception,DNA duplex encoding the -domain (106 bp) of human MT-IA wasconstructed from a chemically-synthesized oligomer by repairsynthesis and enzymatic ligation and cloned into pUC19. Thegenes cloned were sequenced and found to be in the correct orderas designed. Synthetic directional adapters were attached tothe terminals of the -domain gene fragment of human MT-IA toestablish complete control over fragment orientation duringligation. The use of these directional adapters thereby ensuredthe production of multiple copies of the -domain in tandem arrays.The successive -domains were linked by a peptide linker consistingof 10 residues. A chimeric gene containing 12 cloned tandemlyrepeated copies of the 106 bp -domain DNA was introduced intotobacco cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens.A total of 10 different transgenic tobacco plants were generated,of which two showed root and shoot growth unaffected by up to200 mg/l kanamycin and 100 µM cadmium, whereas root growthof control plants was severely inhibited and leaf chlorosisdeveloped on media containing only 10 µM cadmium 相似文献