共查询到20条相似文献,搜索用时 31 毫秒
1.
Pons Jaume; Planas Antoni; Querol Enrique 《Protein engineering, design & selection : PEDS》1995,8(9):939-945
Bacillus 1,3-1,4-ß-glucanases possess a highly conserveddisulfide bridge connecting a ß-strand with a solventexposedloop lying on top of the extended binding site cleft The contributionof the disulfide bond and of both individual cysteines (Cys61and Cys90) in the Bacillus licheniformis enzyme to stabilityand activity has been evaluated by protein engineering methods.Reduction of the disulfide bond has no effect on kinetic parameters,has only a minor effect on the activity-temperature profileat high temperatures, and destabilizes the protein by less than0.7 kcal/mol as measured by equilibrium urea denatu ration at37°C. Replacing either of the Cys residues with Ala destabilizesthe protein and lowers the specific activity. C90A retains 70%of wild-type (wt) activity (in terms of Vmax), whereas C61Aand the double mutant C61AC90A have 10% of wt Vmax. Alarger change in free energy of unfolding is seen by equilibriumurea denaturation for the C61A mutation (loop residue, 3.2 kcal/molrelative to reduced wt) as compared with the C90A mutation (ß-strandresidue, 1.8 kcal/mol relative to reduced wt), while the doublemutant C61AC90A is 0.8 kcal/mol less stable than thesingle C61A mutant. The effects on stability are interpretedas a result of the change in hydrophobic packing that occursupon removal of the sulfur atoms in the Cys to Ala mutations 相似文献
2.
Komeiji Yuto; Uebayasi Masami; Someya Jun-ichiro; Yamato Ichiro 《Protein engineering, design & selection : PEDS》1992,5(8):759-767
The Ser88Cys mutant of the trp-repressor showed a lower affinityfor the corepressor than the wild-type repressor [G = 1.7 ±0.3 kcal/mol, Chou and Matthews (1989) J. Biol. Chem., 264,1831418319].A molecular dynamics/free energy cycle perturbation study wasperformed to understand the origin of the decreased affinity.A value (G = 1.58 ± 0.28 kcal/mol) comparable with theexperimental value was obtained by the simulation. Free energycomponent analysis revealed that destabilization of the vander Waals interaction between Ser88 and Trp109 (corepressor)mainly contributed to the decreased affinity of the mutant.The rotational transition of the hydroxyl (sulfhydryl) groupof Ser88 (Cys88) during the simulations affected the contributionsof Arg84 and water to the free energy change in the aporepressorand those of Arg84 and Trp 109 to that in the holorepressor.However, the contributions from different residues compensatedeach other, and the total free energy changes were almost invariablein the various simulations. 相似文献
3.
Free energy simulations have been employed to rationalize thebinding differences between A-74704, a pseudo C2- symmetricinhibitor of HIV-1 protease and its diester analog. The diesteranalog inhibitor, which misses two hydrogen bonds with the enzymeactive site, is surprisingly only 10-fold weaker. The calculatedfree energy difference of 1.7 ± 0.6 kcal/mol is in agreementwith the experimental result. Further, the simulations showthat such a small difference in binding free energies is dueto (1) weaker hydrogen bond interactions between the two (P1and P1) NH groups of A-74704 with Gly27/Gly27' carbonyls ofthe enzyme and (2) the higher desolvation free energy of A-74704compared with its ester analog. The results of these calculationsand their implications for design of HIV-1 protease inhibitorsare discussed. 相似文献
4.
McDonald Shawn M.; Willson Richard C.; McCammon J.Andrew 《Protein engineering, design & selection : PEDS》1995,8(9):915-924
The titration behavior of the ionizable residues of the HyHEL-5henegg lysozyme complex and its individual components has beenstudied using continuum electrostatic calculations. Severalresidues of HyHEL-5 had pKa values shifted away from model valuesfor isolated residues by more than three pH units. Shifts awayfrom the model values were smaller for the residues of hen egglysozyme. A moderate variation in the pKa values of the titratablegroups was observed upon increase of the ionic strength from0 to 100 mM, amounting to 12 pH units in most cases.Under physiological conditions, the net charge of HyHEL-5 wasopposite that for hen egg lysozyme. Several residues, includingthose involved in the ArgGlu salt bridges that have beenproposed to be important in antibody-antigen binding, had pKavalues that were changed significantly upon binding. The maintitration event upon antibody-antigen binding appears to beloss of a proton from residue GluH50 of the Fv molecule. Thelimitations of our calculation methods and the role they mightplay in the design of antibodies for use in assays, sensorsand separations are discussed 相似文献
5.
Sansom Clare E.; Wu Jin; Weber Irene T. 《Protein engineering, design & selection : PEDS》1992,5(7):659-667
Crystallographic structures of HIV protease with three differentpeptide-mimetic inhibitors were subjected to energy minimizationusing molecular mechanics, the minimized structures analyzedand the inhibitor binding energies calculated. Partial chargeassignment for the hydrogen bonded catalytic aspartk acids,Asp25 and -25', was in good agreement with charge calculationsusing semi-empirical molecular orbital methods. Root mean squaredeviations on minimization were small and similar for both subunitsin the protease dimer. The surface loops, which had the largestB factors, changed most on minimization; the hydrophobic coreand the inhibitor binding site showed little change. The distance-dependentdielectric of D(r) = 4r was found to be preferable to D(r) =r. Distance restraints were applied for the intermolecular hydrogenbonds to maintain the conformation of the inhibitor bindingsite. Using the dielectric of D(r) = 4r, the calculated interactionenergy of the three inhibitors with the protease ranged from53 to 56 kcal/mol. The groups of the inhibitorswere changed to add or remove a transition state analoguehydroxyl group, and the loss in energy on the removal of thisgroup was calculated to be 0.91.7 kcal/mol. This wouldrepresent 1936% of the total measured difference in bindingenergy between the inhibitors JG365 and MVT-101. 相似文献
6.
Ueda Tadashi; Tamura Tomohiro; Maeda Yoshitake; Hashimoto Yoshio; Miki Takeyoshi; Yamada Hidenori; Imoto Taiji 《Protein engineering, design & selection : PEDS》1993,6(2):183-187
Three mutant lysozymes where the Asp101 Gly102 sequenceof lysozyme was converted to Asp101Pro102, Gly101Pro102and Pro101Gly102 were prepared to investigate the effectof proline residues on the stabilization of proteins. The freeenergy changes of lysozymes for the unfolding in aqueous solutionat pH 5.5 and 35°C were 10.0, 10.1, 11.0 and 7.7 kcal/molfor wild type, Asp101Pro102, Gly101Pro102 and Pro101Gly102 lysozymerespectively. When the energy level in the unfolded state ofwild type lysozyme was fixed at a standard level, the energylevels in the folded state of Asp101Pro102 and Pro101Gly102lysozymes were found to be higher than that of wild type lysozymeon the basis of GD(H2O) and entropy losses of their polypeptidechains in the unfolded state. The presence of some strain inthe folded state of these lysozymes was supported by both thecalculation of conformational energy for a trans-L-prolyl residue[Schimmel, P.R. and Flory,P.J. (1968) J. Mol. Biol, 34, 105120] and the analysis of structures of energy-minimizedmutant lysozymes. Therefore, it is concluded that the formationof the Gly-Pro sequence is effective in avoiding possible strainin the folded state of a protein caused by the introductionof proline residue(s). 相似文献
7.
Jimenez M.A.; Arnold J.R.P.; Andrews J.; Thomas J.A.; Roberts G.C.K.; Birdsall B.; Feeney J. 《Protein engineering, design & selection : PEDS》1989,2(8):627-631
The complex of Lactobacillus casei dihydrofolate reductase withthe substrate folate and the coenzyme NADP* has been shown toexist in solution as a mixture of three slowly interconvertingconformations whose proportions are pH-dependent and which differin the orientation of the pteridine ring of the substrate inthe binding site. The Asp26 Asn mutant of L. casei dihydrofolatereductase has been prepared by oligonucleotide-directed mutagenesisand studied by one-and two-dimensional 1H-NMR spectroscopy.NMR studies of the mutant enzymefolateNADP* complexshow that this exists to > 90% in a single conformation overthe pH* range 57.1. The single conformation observedcorresponds to conformation I (the methotrexate-likeconformation) of the wild-type enzymefolateNADP*complex. These observations demonstrate that Asp26 is the ionizablegroup controlling the pH-dependence of the conformational equilibriumseen in the wild-type enzyme. 相似文献
8.
Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants 总被引:5,自引:0,他引:5
Young Robert J.; Owens Raymond J.; Mackay Graham A.; Chan Christina M. W.; Shi Jianguo; Hide Michihiro; Francis David M.; Henry Alistair J.; Sutton Brian J.; Gould Hannah J. 《Protein engineering, design & selection : PEDS》1995,8(2):193-199
We have constructed an expression vector that leads to secretionof the whole Fc of human immunoglobulin E (hIgE-Fc) from mammaliancells at levels up to 100 mg/l of culture. Two surface glycosylationsites at Asn265 and Asn371 have been changed to glutamine, toobtain a more homogeneous preparation of hIgE-Fc for structuralstudies. Comparison of wild-type and mutant products revealedthat Asn371 is rarely glycosylated in Chinese hamster ovarycells. Both the double mutant and wild-type hIgEFc bind to thehigh-affinity IgE receptor, FcRI, with about the same affinityas myeloma IgE (Ka in the range 10101011 M1),and were able to sensitize isolated human basophils for anti-IgEtriggering of histamine release. However, only the double mutanthIgE-Fc approached the affinity of myeloma IgE for the low-affinityreceptor, FcRII (Ka = 7.3x107 M1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (Ka = 4.1x106M1). 相似文献
9.
Mutational analysis of the N-capping box of the {alpha}-helix of chymotrypsin inhibitor 2 总被引:3,自引:0,他引:3
The N-terminus of the helix of the chymotrypsin inhibitor 2from barley (CI2) has an N-capping box (Ser at the first positionin the helix and Glu at position 4) as well as a frequentlyfound Glu at position 3. The energetic importance of this motifhas been studied by determining the free energy of unfoldingof the wild-type and protein mutants derived from those residuesusing guanidinium chloride-induced denaturation and differentialscanning microcalorimetry. Mutating N-cap residue Ser31 to eitherAla or Gly destabilizes CI2 by 0.8-1 kcal mol1. Truncationof the box in the mutants SA31EA33EA34 or SG31EA33EA34 destabilizesthe protein by 1.52 kcal mol1. The N-capping boxis an important motif in stabilizing proteins and delineatingthe beginning of -helices in the pathway of protein folding. 相似文献
10.
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
11.
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
12.
Extrapolation to water of kinetic and equilibrium data for the unfolding of barnase in urea solutions 总被引:5,自引:0,他引:5
Matouschek Andreas; Matthews Jacqueline M.; Johnson Christopher M.; Fersht Alan R. 《Protein engineering, design & selection : PEDS》1994,7(9):1089-1095
Assumptions about the dependence of protein unfolding on theconcentration of urea have been examined by an extensive surveyof the equilibrium unfolding of barnase and many of its mutantsmeasured by urea denaturation and differential scanning calorimetry.The free energy of equilibrium unfolding and the activationenergy for the kinetics of unfolding of proteins are generallyassumed to change linearly with [urea]. A slight downward curvatureis detected, however, in plots of highly precise measurementsof logjtu versus [urea] (where ku is the observed rate constantfor the unfolding of barnase). The data fit the equation logkku= logkuH2O* + mku*.[urea] 0.014[urea]2, where mku*is a variable which depends on the mutation. The constant 0.014 was measured directly on four destabilized mutants and wildtype, and was also determined from a global analysis of data from>60 mutants of barnase. Any equivalent deviations from linearityin the equilibrium unfolding are small and in the same region,as determined from measurements on 166 mutants. The free energyof unfolding of barnase, GUF, appears significantly largerby 1.6 kcal mol1 when measured by calorimetry than whendetermined by urea denaturation. However, the changes in GUFon mutation, GUF, determined by calorimetry and by ureadenaturation are identical. We show analytically how, hi general,the curvature in plots of activation or equilibrium energiesagainst [denaturant] should not affect the changes of thesevalues on mutation provided measurements are made over the sameconcentration ranges of denaturant and the curvature is independentof mutation. 相似文献
13.
The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability 总被引:8,自引:0,他引:8
Zhou Nian E.; Kay Cyril M.; Hodges Robert S. 《Protein engineering, design & selection : PEDS》1994,7(11):1365-1372
The net energetic contribution of interhelical electrostaticattractions to coiled-coil stability has been quantitated usingde novo designed synthetic coiled-coils. The synthesized modelcoiled-coil (EK), denoted by amino acid res-idues in positionse and g, which contains only interhelical ionic interactionswithout any possible (i, i + 3) and (i, i + 4) intrahelicalionic interaction, consists of two identical 35 residue polypeptidechains with a heptad repeat KgLaG-bAcLdEeKf. Three mutant coiled-coilswere prepared where five Glu residues at e positions in EK weremutated to Gin residues (QK); five Lys residues at g positionswere altered to Gin residues (EQ) or these mutations were effectedat both positions e and g (QQ). The stabilities of the fourcoiled-coils were determined by measuring the ellipticitiesat 220 nm as a function of urea concentration at 20C. By usinga double-mutant cycle analysis it was possible to isolate theenergetic contribution of interhelical ionic attractions tocoiled-coil stability from the other contributions such as helicalpreference and hydro-phobicity. The 0.37 0.01 kcal/mol ofenergetic contribution of one interhelical ion pair to the coiled-coilstability was obtained from three independent comparisons. Thisfinding suggests that a large number of weak interhelical electrostaticinteractions on the surface of a protein can make a substantialcontribution to protein stability. In addition, the energeticcontributions of a single mutation E Q, K+Q, Q E andE Ewere also determined (G = 0.22, 0.26, 0.46 and 0.65kcal/mol for the single mutations, respectively). The greatercontribution of a protonated Glu residue to coiled-coil stabilitycompared with an ionized Glu residue (0.65 kcal/mol) can outweighthe relatively smaller contribution of an interhelical ion pair(0.37 kcal/mol), which clearly explains why most coiled-coilsare more stable at acidic pH compared with neutral pH even wheninterhelical salt bridges contribute to the coiled-coil stabilityat neutral pH. 相似文献
14.
We present free energy perturbation calculations on the complexesof Glu46 Ala46 (E46A) and Glu46 Gln46 (E46Q) mutantsof ribonuclease T1 (RNaseT1) with inhibitors 2-guanosinemonophosphate (GMP) and 2adenosine monophosphate (AMP)by a thermodynamic perturbation method implemented with moleculardynamics (MD). Using the available crystal structure of theRNaseT1GMP complex, the structures of E46A-GMP and E46Q-GMPwere model built and equilibrated with MD simulations. The structuresof E46A-AMP and E46Q-AMP were obtained as a final structureof the GMPAMP perturbation calculation respectively.The calculated difference in the free energy of binding (Gbind)was 0.31 kcal/mol for the E46A system and 1.04 kcal/molfor the E46Q system. The resultant free energies are much smallerthan the experimental and calculated value of 3 kcal/mol forthe native RNase T1, which suggests that both mutants have greaterrelative adenine affinities than native RNaseT1. EspeciallyE46Q is calculated to have a larger affinity for adenine thanguanine, as we suggested previously from the calculation onthe native RNaseT1. Thus, the molecular dynamics/free energyperturbation method may be helpful in protein engineering, directedtoward increasing or changing the substrate specificity of enzymes. 相似文献
15.
McDowall Sharon; Heeswijck Robyn van; Hoogenraad Nicholas 《Protein engineering, design & selection : PEDS》1990,4(1):73-77
We have investigated the putative carbamylphosphate- and ornithine-bindingdomains in ornithine transcarbamylase from rat liver using site-directedmutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-Xand therefore implicated in the binding of the phosphate moietyof carbamylphosphate has been replaced with a leucine. Thisresults in a dramatic reduction of catalytic activity, althoughthe enzyme is synthesized in cells stably transfected with themutant clone and imported, correctly processed and assembledinto a homotrimer in mitochondria. The sole cysteine residue(Cys271) has been implicated in ornithine binding by the chemicalmodification studies of Marshall and Cohen in 1972 and 1980(J. Biol. Chem., 247, 16541668, 16691682; 255,72917295, 72967300). Replacement of this residuewith serine did not eliminate enzyme activity but affected theMichaelis constant for ornithine (Kb, increasing it 5-fold from0.71 to 3.7 mM and reduced the kcat at pH 8.5 by 20-fold. Thesechanges represent a loss in apparent binding energy for theenzyme - ornithine complex of 2.9 kcal/mol, suggesting thatCys271 is normally involved in hydrogen bonding to the substrate,ornithine. The cysteine to serine substitution also caused thedissociation constant (Kä for the competitive inhibitor,L-norvaline to be increased 10-fold, from 12 to 120 µM.The small loss in binding energy and relatively high residualcatalytic activity of the mutant strongly suggests that a numberof other residues are involved in the binding of ornithine.The effect of replacement of Cys271 with serine was restrictedto the ornithine binding site of the enzyme since both the bindingconstant for carbamyl-phosphate (Kia) and Michaelis constant(Ka) were not appreciably different for mutant and wild-typeenzymes. The pH optimum of the wild-type enzyme (8.6) is increasedto > 9.6 in the Ser271 mutant. 相似文献
16.
Free energy simulations (slow-change method) have been usedto estimate quantitatively the ratio of the binding constantsof (S) and (R) isomers of a novel HIV protease inhibitor, JG365.As a starting geometry, we used the X-ray crystallographic structureof a complex of HTV protease and JG365 provided by A.Wlodawer.According to our results the (S) configuration, i.e. the formpreviously identified experimentally, binds considerably moretightly to the protease (G° = 2.9 kcal/mol). When the (S)inhibitor is bound, there is a very strong preference for protonationof the Aspl25 (rather than the Asp25) residue of the protease.This study is the first to apply a new method for quantitativelyassessing the precision of free energies calculated by the slow-changemethod 相似文献
17.
The binding of the La3+ ion to a tridecapeptide, which is amodel for the EF-hand in calcium-binding proteins, is studiedhi solution by free energy simulations. The calculations analyzethe effect on the La3+ ion binding of the mutation of Asp toAsn for side chains that interact directly with the ion. Theresults are compared with the measurements of Marsden.B.J.,Hodges, R.S. and Sykes, B.D. (1989) Biochemistry, 28,8839, onthe same system. They found that the Asp to Asn mutation hasonly a small effect on the binding; the observed differencesin the free energies on changing one Asp to an Asn are between-0.3 and 1.8 kcal/ mol. This result is analyzed by alchemicalsimulations for the tridecapeptide in the bound Qoop) structureand free (extended) form. The free energy changes due to themutation of an Asp to an Asn are large and positive for boththe bound and free forms. However, since the values of the freeenergy changes are calculated to be similar hi the two forms,the difference in the binding free energy of Asp and Asn peptidesis found to be small, in agreement with experiment. By use ofthermodynamic integration, the various contributions to thefree energy changes are estimated. In the com-plexed form, theAsp to Asn mutation is favored by the reduction in the repulsiveinteraction with other charged residues of the peptide; it isdisfavored by the reduction of the stabilization of the ionand the surrounding water has a small effect. When the peptideadopts an extended conformation in the absence of the ion, themutation Asp to Asn is strongly disfavored by the interactionswith the water and is favored by the interactions within thepeptide. The results demonstrate the essential role of contributionsto the binding of EF-hands from interactions other than thosebetween the ion and the charged amino acid side chains. Theresults obtained from the simulations suggest, in accord withcrystal structures of La3+ bound to various ligands, that thecalcium-binding loop complexed with La3+ in solution has a significantlydifferent structure from that observed hi proteins. 相似文献
18.
Imoto Taiji; Yamada Hidenori; Yasukochi Takanori; Yamada Eiichi; Ito Yuji; Ueda Tadashi; Nagatani Hiroko; Miki Takeyoshi; Horiuchi Tadao 《Protein engineering, design & selection : PEDS》1987,1(4):333-338
In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala31ValandAsn106Ser)in addition to an extra methionine residue at theNH2-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met1 Val31Ser106-, Met1Ser106-,Met1 Val31-and Met1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH2-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn106 by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala31Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme. 相似文献
19.
Synthetic genes (A, AB and AHB) constructed and cloned intopKK233-2 vector were recloned from the parent plasmid into thenew procaryotic expression vectors pGFY221N and pBIO52. GeneAFB (coding for all amino acids besides phenylalanine)was obtained by cassette mutagenesis from geneAB. The plasmid pGFY221N was constructed from pGFY218L by replacingthe PstI by an NcoI site; plasmid pBIO52 was derived from pGFY221Nthrough replacing the 221-bp EoRl/NcoI fragment with a syntheticDNA segment of 52 bp representing the Escherichia coli atpEgene translational initiation region. The genes A, AB, AHB andAFB in the vector pGFY221N were expressed with a six-amino-acid-longleader sequence; in pBIO52 the genes were expressed directly.in vitro expression experiments were successful with all thegenes except with the AHB gene integrated into pGFY221N. Inthe E. coli minicell system expression was demonstrated withthe A gene in pGFY221N and the AFB and AHB genes in pBIO52.Complete translation of the expressed genes AB, AFB andAHB in either the in vitro or in vivo systems could be shownby using 35S-labelled N-terminal methionine and C-terminal cysteine.Both amino acids occur only once in the peptide sequences. 相似文献
20.
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