共查询到20条相似文献,搜索用时 15 毫秒
1.
Smallshaw Joan E.; Georges Fawzy; Lee Jeremy S.; Waygood E.Bruce 《Protein engineering, design & selection : PEDS》1999,12(7):623-630
The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptideheavy chainlinkerlightchain(His)5 tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 1030% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with Kd values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay. 相似文献
2.
3.
Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps 总被引:1,自引:0,他引:1
Gish Gerald; McGlone Maria L.; Pawson Tony; Adams Joseph A. 《Protein engineering, design & selection : PEDS》1995,8(6):609-614
The gene coding for the tyrosine protein kinase domain of v-fpswas subcloned into a plasmid vector expressing glutathione-S-transferase(GST). This new vector expresses a fusion protein in Escherichiacoli composed of the kinase domain linked with GST at the N-terminus(GST-kin). A portion of the total expressed protein was solubleupon cell lysis and was purified by affinity chromatographyusing glutathione cross-linked agarose. GST-kin (Mr 57 000)is a phosphoprotein as judged by 32P autoradiography, consistentwith the known autophosphorylation site within the kinase core[Weinmaster et aL (1984) Cell, 37, 559568]. Cleavageof the fusion protein with thrombin and purification on phosphocelluloseresin yielded the pure kinase domain (Mr 33 000). The activityof the kinase domain is indistinguishable from that of GST-kinusing the peptide substrate EEEIYEEIE, indicating that Nterminalfusion has no effect on the kinase domain. GSTkin phosphorylatesa second peptide, EAEIYEAIE, with improved catalytic efficiency.Initial velocity data are consistent with a random bireactantmechanism with no substrate synergism observed in the ternarycomplex. Steady-state kinetic analyses reveal that this peptideis phosphorylated, with a kcat of 3.6 s1, a Kpeptideof 500 µM and a KATP of 250 µM. The expression,purification and preliminary kinetic analysis of the kinasedomain of v-fps provide the first step in the application ofstructurefunction studies for this oncoprotein 相似文献
4.
Degryse E.; Acker M.; Defreyn G.; Bernat A.; Maffrand J.P.; Roitsch C.; Courtney M. 《Protein engineering, design & selection : PEDS》1989,2(6):459-465
The hirudin variant HV2 was modified by in vitro site-specificmutagenesis of HV2 cDNA to generate HV2(Asn-47 Lys),HV2(Asn-47 Arg) and HV2(Lys-35 Thr, Asn-47 Lys). Residues 35 and 47 are positioned respectively withinthe finger and prothrombin-like domains of hirudin, both ofwhich have been suggested as thrombin binding sites. The modifiedpolypeptides were synthesized in Saccharomyces cerevisiae usinga secretion vector and purified from culture supernatants. Byanalysis of the human -thrombin: hirudin inhibition reactionin steady-state conditions it was shown that the dissociationconstants for HV2(Lys-47) and HV2(Arg-47) were 5- to 14-foldlower than for unmodified HV2, whereas mutation of Lys-35 didnot significantly alter the inhibition kinetics. Furthermore,HV2(Lys-47), whose sequence is identical to a natural hirudinvariant, displayed enhanced antithrombotic activity in vivo,having a 100-fold lower ED50 compared to HV2 in the rabbit Wesslervenous thrombosis model. These results support a role for theprothrombin-like domain in thrombin binding and, moreover, demonstratethat in vivo antithrombotic efficiency correlates with the dissociationconstant of the inhibition reaction. 相似文献
5.
Peter W.; Dunkel R.; Stouten P.F.W.; Vriend G.; Beato M.; Suske G. 《Protein engineering, design & selection : PEDS》1992,5(4):351-359
The progesteronebinding protein uteroglobin has beenexpressed in Escherichia coli in an unfused, soluble form. likemature uteroglobin from rabbit endometrium (UG), the E.coliproduceduteroglobin (UG1) dimerizes in vitro, forms an antiparalleldimer with Cys3Cys69' and Cys69Cys3' disulfidebonds and binds progesterone under reducing conditions. In orderto analyze the dimerization and the reduction dependence ofprogesterone binding in more detail, we separately replacedcysteine 3 and cysteine 69 by serines. Under reducing conditions,both uteroglobin variants (UGl3Ser and UGl69Ser)bind progesterone with the same affinity as the wildtypesuggesting that both cysteine residues are not directly involvedin progesterone binding. In contrast to the wildtypeprotein, both cysteine variants also bind progesterone withhigh affinity in the absence of reducing agents. In addition,UGl-3Ser and UGl-69Ser both form covalently linked homodimers.Thus, unnatural Cys6969' and Cys33' disulfidebonds exist in UG13Ser and UG169Ser, respectively.These data together with computer models based on X-ray diffractiondata strongly support the idea that progesterone reaches itsbinding site located in an internal hydrophobic cavity via ahydrophobic tunnel along helices 1 and 4. Under nonreducingconditions the tunnel is closed by two disulfide bridges (Cys3Cys69'(and Cys69Cys3') that lie in the most flexible regionof the dimer. Reduction or replacement of a cysteine residueenables conformational changes that open the channel allowingprogesterone to enter. 相似文献
6.
Siezen Roland J.; Bruinenberg Paul G.; Vos Pieter; Alen-Boerrigter Ingrid Van; Nijhuis Monique; Alting Arno C.; Exterkate Fred A.; Vos Willem M.de 《Protein engineering, design & selection : PEDS》1993,6(8):927-937
The substrate-binding region of the cell-envelope proteinaseof Lactococcus lactis strain SK11 was modelled, based on sequencebomology of the catalytic domain with the serine proteinasessubtilisin and thermitase. Substitutions, deletions and insertionswere introduced, by site-directed and cassette mutagenesfe ofthe prtP gene encoding this enzyme, based on sequence comparisonboth with subtilisin and with the homologous L.lactis strainWg2 proteinase, which has different proteolytic properties.The engineered enzymes were investigated for thermal stability,proteolytic activity and cleavage specificity towards smallchromogenk peptide substrates and the peptide g1-casein(l23).Mutations in the subtilisin-like substrate-binding region showedthat Ser433 is the active site residue, and that residues 138and 166 at either side of the binding cleft play an importantrole in substrate specificity, particularly when these residuesand the substrate are oppositely charged. The K748T mutationin a different domain also affected specificity and stability,suggesting that this residue is in close proximity to the subtilisin-likedomain and may form part of the substratebinding site. Severalmutant SK11 proteinases have novel properties not previouslyencountered in natural variants. Replacements of residues 137139AKTalong one side of the binding cleft produced the 137139GPPmutant proteinase with reduced activity and narrowed specificity,and the 137139GLA mutant with increased activity andbroader specificity. Furthermore, the 137139GDT mutanthad a specificity towards g1,-casein(l23) closely resemblingthat of L.lactis Wg2 proteinase. Mutants with an additionalnegative charge in the binding region were more stable towardsautoproteolysis. 相似文献
7.
Bakker Arjen H. F.; Rehberg Edward F.; Marotti Keith R.; Verheijen Jan H. 《Protein engineering, design & selection : PEDS》1995,8(3):293-300
Tissue-type plasminogen activator (t-PA) is composed of structurallyautonomous domains. From the N-terminus of t-PA, a finger-likedomain (F), an epidermal growth factor-like domain (G), twokringle domains (Kl and K2) and a serine protease domain (P)can be discerned. The K2 domain of t-PA is known to be involvedin lysine binding, fibrin binding and fibrin-dependent plasminogenactivation. To study the functional autonomy of the K2 domainin t-PA we constructed, with the aid of a cassette t-PA gene[Rehberg et al. (1989) Protein Engng, 2,371377], mutantt-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2Pand GK2K1P) in which the K2 domain was placed in two differentpositions in t-PA. The DNAs of wild-type t-PA and the t-PA variantswere expressed in Chinese hamster ovary cells and the recombinantproteins were purified by affinity chromatography.All moleculeswere expressed in their single-chain form and could be convertedto their two-chain form. With these molecules, lysine binding,fibrin binding and fibrin-dependent plasminogen activation werestudied. All variants showed affinity for lysyl-Sepharose andaminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versusFGK2K1P and GK1K2P versus GK2K1P) resulted in a 2347%weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose.Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versusGK2K1P) resulted in a 2070% improvement of the interactionslysyl-Sepharose and aminohexyl-Sepharose. All variants boundto a forming fibrin clot. Reversal of the K domains (FGK1K2Pversus FGK2K1P) reduced fibrin binding. In the presence of thelysine analogue -amino caproic acid, only FGK1K2P bound to fibrin.All variants activated plasminogen. In the absence of fibrinogenCNBr fragments (mimic of fibrin), the reversal of the K domain(FGK2K1P) resulted in a 2-fold improved plasminogen activation.In the presence of a fibrin mimic, the plasminogen activationsof the F domain deletion analogues GK1K2P and GK2K1P were foundto be decreased 2- to 4-fold. From these results we concludedthat the function of t-PA in lysine binding, fibrin bindingand fibrin-dependent plasminogen activation is dependent onthe correct spatial orientation of the K2 domain within thet-PA molecule 相似文献
8.
Residues important for the function of a multihelical DNA binding domain in the new transcription factor family of Cam and Tet repressors 总被引:5,自引:0,他引:5
Aramaki Hironori; Yagi Naoto; Suzuki Masashi 《Protein engineering, design & selection : PEDS》1995,8(12):1259-1266
We report that some prokaryotic repressors including CamR andTetR belong to the same family. CamR and TetR bind to DNA usinga multihelical DNA binding domain (DBD) at the N-termini ofthe proteins, while the C-termini are important for regulatingthe DNA binding in a manner dependent on their co-factors (camphorfor CamR, tetracycline for TetR). In all, 11 important aminoacid positions have been identified in the CamR DBD by the systematicsubstitution of residues by Ala. Of the 11 positions, 10 areeither buried in the core, and thus important for creating thehydrophobic environment, or exposed on the surface, and thusimportant for binding to DNA. The eleventh residue, Gly, seemsto be important for a loop structure. The DNA binding mode ofthis type of DBD and a general mechanism of regulating theirDNA binding are discussed in reference to the crystal structureof TetR [Hinrichs et al., (1994) Science, 264, 418420]. 相似文献
9.
Haris Parvez I.; Ramesh Bala; Sansom Mark S. P.; Kerr Ian D.; Srai Kaila S.; Chapman Dennis 《Protein engineering, design & selection : PEDS》1994,7(2):255-262
Recent mutagenesis studies nave identified a stretch of aminoacid residues which form the ion-selective pore of the voltage-gatedpotassium channel. It has been suggested that this sequenceof amino acids forms a ß-barrel structure making upthe structure of the ion-selective pore [Hartman,H.A., Kirsch,G.E.,DreweJ.A., Taglialatela.M., Joho.R.H. and Brown,A.M. (1991)Science, 251, 942944; YeUen.G., Jurman,M.E., Abramson,T.and MacKinnon,R. (1991) Science, 251, 939942; Yool,AJ.and Schwarz.T.L. (1991) Nature, 349, 700704]. We havesynthesized a polypeptide corresponding to this amino add sequence(residues 431449 of the ShA potassium channel from Drosophila).A tetrameric version of this sequence was also synthesized byUnking together four of these peptldes onto a branching lysinecore. Fourier transform infrared (FT-LR) and circular dichroism(CD) spectroscopy have been used to investigate the structureof these peptides after their reconstitution into lyso phos-phatidylcholinemicelles and lipid bilayers composed of dimyristoyl phosphatidyfcholineand dimyristoyl phosphatidyl-glycerol. The spectroscopic studiesshow that these peptides are predominantly a-helical in theselipid environments. When Incorporated into planar lipid bilayersboth peptides induce ion channel activity. Molecular modellingstudies based upon the propensity of these peptides to forman -helical secondary structure in a hydrophobfc environmentare described. These results are discussed in the light of recentmutagenesis and binding studies of the Drosophila Shaker potassiumion channel protein 相似文献
10.
Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences 总被引:3,自引:0,他引:3
Tomizawa Hideyuki; Yamada Hidenori; Hashimoto Yoshio; Imoto Taiji 《Protein engineering, design & selection : PEDS》1995,8(10):1023-1028
Site-directed mutagenesis was performed at Asp-Gly (4849,6667, 101102) and Asn-Gly (103104) sequencesof hen egg-white lysozyme to protect the enzyme against irreversiblethermoinactivation. Because the lysozyme inactivation was causedby the accumulation of multiple chemical reactions, includingthe isomerization of the Asp-Gly sequence and the deamidationof Asn [Tomizawa et al.(1994) Biochemistry, 33, 1303213037],the suppression of these reactions by the substitution of Glyto Ala, or the introduction of a sequence of human-type lysozyme,was attempted and the mutants (where each or all labile sequenceswere replaced) were prepared. The substitution resulted in thereversible destabilization from 1 to 2 kcal/mol per substitution.The destabilization was caused by the introduction of ß-carbonto the constrained position that had conformational angles withinthe allowed range for the Gly residue. Despite the decreasein the reversible conformational stability, the mutants hadmore resistance to irreversible inactivation at pH 4 and 100°C.In particular, the rate of irreversible inactivation of themutant, which was replaced at four chemically labile sequences,was the latest and corresponded to 18 kcal/mol of the reversibleconformational stability. Therefore, replacement of the chemicallylabile sequence was found to be more effective at protectingenzymes against irreversible thermoinactivation than at strengtheningreversible conformational stability. 相似文献
11.
Prediction of the antigenic sites of the cystic fibrosis transmembrane conductance regulator protein by molecular modelling 总被引:1,自引:0,他引:1
Gallet Xavier; Benhabiles Nora; Lewin Miguel; Brasseur Robert; Thomas-Soumarmon Annick 《Protein engineering, design & selection : PEDS》1995,8(8):829-834
Antibodies are powerful tools for studying the in situ localizationand physiology of proteins. The prediction of epitopes by molecularmodelling has been used successfully for the papilloma virus,and valuable antibodies have been raised [Muller et aL (1990)J. Gen. Virol, 71, 27092717]. We have improved the modellingapproach to allow us to predict epitopes from the primary sequencesof the cystic fibrosis transmembrane conductance regulator.The procedure involves searching for fragments of primary sequenceslikely to make amphipathic secondary structures, which are hydrophilicenough to be at the surface of the folded protein and thus accessibleto antibodies. Amphipathic helices were predicted using themethods of Berzofsky, Eisenberg and Jahnig. Their hydrophobichydrophilicinterface was calculated and drawn, and used to predict theorientation of the helices at the surface of the native protein.Amino acids involved in turns were selected using the algorithmof Eisenberg. Tertiary structures were calculated using FOLDING,a software developed by R.Brasseur for the prediction of smallprotein structures [Brasseur (1995) J. MoL Graphics, in press].We selected sequences that folded as turns with at least fiveprotruding polar residues. One important property of antibodiesis selectivity. To optimize the selectivity of the raised antibodies,each sequence was screened for similarity (FASTA) to the proteinsequences from several databanks. Ubiquitous sequences werediscarded. This approach led to the identification of 13 potentialepitopes in the cystic fibrosis transmembrane conductance regulator:seven helices and six loops. 相似文献
12.
1.85 A structure of anti-fluorescein 4-4-20 Fab 总被引:1,自引:0,他引:1
Whitlow Marc; Howard Andrew J.; Wood Jay F.; Voss Edward W. Jr; Hardman Karl D. 《Protein engineering, design & selection : PEDS》1995,8(8):749-761
The crystal complex of fluorescein bound to the high-affinityanti-fluorescein 4-4-20 Fab {Ka = 1010 M1 at 2°C)has been determined at 1.85 Å. Isomorphous crystals oftwo isoelectric forms (p1 = 7.5 and 7.9) of the antifluorescein4-4-20 Fab, an IgG2A [Gibson et al (1988)Proteins: Struct. FunctGenet., 3, 155160], have been grown. Both complexes crystallizewith one molecule in the asymmetric unit in space group P1,with a = 42.75 Å, b =43.87 Å, c = 58.17 Å, = 95.15° , ß = 86.85° and = 98.01°.The final structure has an R value of 0.188 at 1.85 Åresolution. Interactions between bound fluorescein, the complementarity-determiningregions (CDRs) of the Fab and the active-site mutants of the4-4-20 single-chain Fv will be discussed. Differences were foundbetween the structure reported here and the previously reported2.7 Å 4-4-20 Fab structure [Herron et al. (1989) Proteins:Struct. Fund., 5, 271280]. Our structure determinationwas based on 26 328 unique reflections four times theamount of data used in the previous report. Differences in thetwo structures could be explained by differences in interpretingthe electron density maps at the various resolutions. The r.m.s.deviations between the variable and constant domains of thetwo structures were 0.77 and 1.54 Å, respectively. Fourregions of the light chain and four regions of the heavy chainhad r.m.s. backbone deviations of >4 Å. The most significantof these was the conformation of the light chain CDR 1. 相似文献
13.
Display of {beta}-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-{beta}-lactamase fusions 总被引:3,自引:0,他引:3
Georgiou George; Stephens Daren L.; Stathopoulos Christos; Poetschke Heather L.; Mendenhall John; Earhart Charles F. 《Protein engineering, design & selection : PEDS》1996,9(2):239-247
Bacterial cell-surface exposure of foreign peptides and solubleproteins has been achieved recently by employing a fusion proteinmethodology. An Lpp'OmpA(46159)Bla fusionprotein has been shown previously to display the normally periplasmicenzyme ß-lactamase (Bla) on the cell surface of theGram-negative bacterium Escherichia coli. Here, we have investigatedthe role of the OmpA domain of the tripartite fusion proteinin the surface display of the passenger domain (Bla) and havecharacterized the effects of the fusion proteins on the integrityand permeability of the outer membrane. We show that in additionto OmpA(46159), a second OmpA segment, consisting ofamino acids 4666, can also mediate the display of Blaon the cell surface. Other OmpA domains of various lengths (aminoacids 4684, 46109, 46128, 46141and 46145) either anchored the Bla domain on the periplasmicface of the outer membrane or caused a major disruption of theouter membrane, allowing the penetration of antibodies intothe cell. Detergent and antibiotic sensitivity and periplasmicleakage assays showed that changes in the permeability of theouter membrane are an unavoidable consequence of displayinga large periplasmic protein on the surface of E.coli. This isthe first systematic report on the effects that cell surfaceengineering may have on the integrity and permeability propertiesof bacterial outer membranes. 相似文献
14.
Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex 总被引:1,自引:0,他引:1
Molecular recognition and site-directed mutagenesis are usedin combination to identify kinetically, transition state interactionsbetween glucoamylase (GA) and the substrate maltose. Earlierstudies of mutant Glu180 Gin GA had indicated a rolein substrate binding for Glul80 (Slerks, M.R., Ford, C., Reilly,P.J. and Svensson, B. (1990) Protein Engng, 3, 193198).Here, changes in activation energies calculated from measuredkcat/Km values for a series of deoxygenated maltose analoguesindicate hydrogen bonding between the mutant enzyme and the3-OH group of the reducing end sugar ring. Using the same substrateanalogues and determining activation energies with wild-typeGA an additional hydrogen bond with the 2-OH group of maltoseis attributed to an interaction with the carboxylate Glu180.This novel combination of molecular recognition and site-directedmutagenesis enables an enzyme substrate transition state contactto be identified and characterized even without access to thethree dimensional structure of the enzyme. Given the distantstructural relationships between glucoamylases and several starchhydrolases (Svensson, B. (1988) FEBS Lett., 230, 7276),such identified contacts may ultimately guide tailoring of theactivity of these related enzymes. 相似文献
15.
Rypniewski W.R.; Hastrup S.; Betzel Ch.; Dauter M.; Dauter Z.; Papendorf G.; Branner S.; Wilson K.S. 《Protein engineering, design & selection : PEDS》1993,6(4):341-348
The trypsin from Fusarium oxysporum is equally homologous totrypsins from Streptomyces griseus, Streptomyces erythraeusand to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibitedform of the enzyme has been crystallized from 1.4 M Na2SO4,buffered with citrate at pH 5.05.5. The crystals belongto space group P21 with cell parameters a=33.43 Å, b=67.65Å, c=39.85 Å and ß=107.6°. There isone protein molecule in the asymmetric unit. X-ray diffractiondata to a resolution of 1.8 Å were collected on film usingsynchrotron radiation. The structure was solved by molecularreplacement using models of bovine and S.griseus trypsins andrefined to an R-factor of 0.141. The overall fold is similarto other trypsins, with some insertions and deletions. Thereis no evidence of the divalent cation binding sites seen inother trypsins. The covalently bound inhibitor molecule is clearlyvisible. 相似文献
16.
Francescutti D.; Baldwin J.; Lee L.; Mutus B. 《Protein engineering, design & selection : PEDS》1996,9(2):189-194
The catalytic properties of glutathione reductase for its substrate,glutathione disulfide, were altered following a 60 s exposureto a 100-fold molar excess of peroxynitrite; the KM value wasincreased by {small tilde}2.5-fold and the Vmax value was decreasedby {small tilde}1.7-fold. The kinetic alterations are thoughtto result from nitrotyrosine formation as the intrinsic Tyrfluorescence is diminished. The UV-visible spectrum of glutathionereductase exhibited absorbance at {small tilde}423 nm, characteristicof nitrotyrosine. In addition, the presence of nitrotyrosinehas been detected by Western immunoblots with an anti-nitrotyrosineantibody. The peroxynitrite-induced inactivation is not observedin the presence of excess glutathione disulfide. However, excessNADPH offered no protection against peroxynitriteinduced inactivation.These observations suggest that the modification of {small tilde}1.8Tyr per subunit, at or near the glutathione disulfide bindingdomain, probably results in the observed catalytic alterations.To test this hypothesis, the two tyrosines closest to the glutathionedisulfide binding domain (Tyr114 and Tyr106), as indicated bythe X-ray crystallographic data [Karplus and Schulz (1989) J.Biol. Chem, 210,163180], were each converted to nitrotyrosinesby molecular modeling and the structure energy was minimized.These theoretical calculations indicate that the bond lengthsbetween Tyr114-O and the Gly-N and Cys II-N of glutathione disulfidebound to glutathione reductase (Karplus and Schulz, 1989) increasedby 3.0 and 4.3 Å, respectively, upon nitration. In thecase of Tyr106, the OCys II-N distance also increasesby {small tilde}1.6 Å. The loss of these hydrogen bondingcontacts is likely to result in the observed catalytic alterationsupon reaction with peroxy-nitrite. 相似文献
17.
Site-specific mutagenesis was employed to study structure-functionrelationships at the substrate binding site of rat tissue kallikrein.Four kallikrein mutants, the Pro219 deletion (P219del), the3438 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(3438)IN],the Trp215Gly exchange (W215G) and the double mutant with Tyr99Hisand Trp215Gly exchange (Y99H:W215G) were created by site-directedmutagenesis to probe their function in substrate binding. Themutant proteins were expressed in Esclzerichia coli at highlevels and analyzed by Western blot. These mutant enzymes werepurified to apparent homogeneity. Each migrated as a singleband on SDS-PAGE, with slightly lower molecular mass (36 kDa)than that of the native enzyme, (38 kDa) because of their lackof glycosylation. The recombinant kallikreins are immunologicallyidentical to the native enzyme, displaying parallelism withthe native enzyme in a direct radioimmunoassay for rat tissuekallikrein. Kinetic analyses of Km and kcat using fluorogenicpeptide substrates support the hypothesis that the Tyr99Trp215interaction is a major determinant for hydrophobic P2 specificity.The results suggest an important role for the 3438 loopin hydrophobic P3 affinity and further show that Pro219 is essentialto substrate binding and efficient catalysis of tissue kallikrein. 相似文献
18.
Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module 总被引:1,自引:0,他引:1
Riley L.G.; Ralston G.B.; Weiss A.S. 《Protein engineering, design & selection : PEDS》1996,9(2):223-230
Human c-Jun and c-Fos leucine zipper domains were examined fortheir ability to serve as autonomous dimerization domains aspart of a heterologous protein construct. Schistosoma japonicumglutathione S-transferase (GST) was fused to recombinant Junleucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains.SDSPAGE snapshot analyses based on disulphidelinkage of monomers demonstrated the ability of rJunLZ to functionas a dimerization motif in a foreign protein environment. Sterichindrance prevented formation of rJunLZGST::rFosLZGSTheterodimers whereas rJunLZGST::rFosLZ and rJunLZ::rFosLZGSTformed readily. Furthermore, rJunLZGST generated homodimerssuggesting fusion protein heterodimers interact differentlyto homodimers. Gel filtration chromatography confirmed thatGST is a dimer in solution and that attachment of a leucinezipper domain allows further interactions to take place. Sedimentationequilibrium analyses showed that GST is a stable dimer (Ka >106 M-1) with no higher multimeric forms. rFosLZGST weaklyassociates beyond a dimer (Ka {small tilde}4x105 M-1) and rJunLZGSTassociates indefinitely (Ka {small tilde}4x106 M-1), consistentwith an isodesmic model of association. The interaction of theseleucine zippers independently of GST association demonstratestheir utility in the modification of proteins when multimerformation is desired. 相似文献
19.
Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis 总被引:3,自引:0,他引:3
One approach to the analysis of leucocyte cell surface proteinsis to express their domains with part of another protein asa carrier. We report the use of two immunoglobulin superfamily(IgSF) domains from rat CD4 (CD4d3+4) in producing domains fromvarious superfamilies as chimeric proteins in Chinese hamsterovary cell lines. Four types of construct were successfullyexpressed containing: (i) the two IgSF domains of CD48; (ii)the IgSF domain of mb-1 which is part of the B cell antigenrecognition complex; (iii) a T cell receptor V domain; and (iv)the N-terminal domain of CD5 which belongs to the scavengerreceptor superfamily. This CD5 chimeric protein was antigenkfor a panel of CD5 mAbs showing that mAbs with functional effectsreacted with the N-terminal domain of CD5. The CD48 chimericprotein has been used both as multivalent complexes producedby crosslinking with mAbs recognizing CD4 and in a monomericform to analyse the kinetics of the interaction between CD48and CD2 [van der Merwe et al. (1993) EMBO J., 12, 49454954]. 相似文献
20.
Coutinho Pedro M.; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1994,7(3):393-400
Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 68 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains. 相似文献