共查询到20条相似文献,搜索用时 15 毫秒
1.
Stocklin Reto; Green Brian N.; Offord Robin E. 《Protein engineering, design & selection : PEDS》1994,7(2):285-287
Insulin analogues labelled with stable isotopes (e.g. deuterium,18O, 15N, etc.) are authentic (the native structure is rigorouslymaintained), non-radioactive (preferred for injection into man)and can easily be distinguished from endogenous insulin by massspectrometry by virtue of their molecular masses. Appropriatecombinations of amino-protecting groups (methylsulphonylethyloxycarbonyland t-butoxy carbonyl), Edman degradation and chemical couplingwere used to produce [octadeutero-PheB1]-porcine insulin and[octadeutero-PheB1-octadeutero-ValB2]-porcine insulin. The analogueswere characterized by electrospray ionization mass spectrometry.Standard mixtures of labelled and unlabelled insulins were successfullystudied by mass spectrometry. Isotope dilution mass spectrometrycould therefore provide a useful direct measure of insulin undertrue physiological conditions, without many of the drawbacksof existing methods. In this regard, the analogue with 16 deuteriumswas more suitable than the octadeuterated analogue, since thegreater mass difference between the labelled and unlabelledforms enabled a lower mass spectrometric resolution to be used,resulting in higher sensitivity 相似文献
2.
Rose Keith; Stocklin Reto; Savoy Luc-Alain; Regamey Pierre-Olivier; Offord Robin E.; Vuagnat Pierre; Markussen Jan 《Protein engineering, design & selection : PEDS》1991,4(4):409-412
The production of semisynthetic human insulin for therapeuticpurposes is of considerable importance. During trypsin-catalysedtransformation of pig insulin into an ester of insulin of humansequence, the alanyl residue at position B30 is removed andreplaced with an esterified residue of threonine. We have carriedout this transformation in a medium enriched in 18OH2 and studiedthe product by MS. In contrast to a previous report, we findthat incorporation of label into the B29B30 peptide bondoccurs during the transformation with threonine methyl esterin aqueous N, N-dimethylacetamide. Quantitative data are presentedand the implications of these findings are discussed. 相似文献
3.
The subunit structure of human macrophage migration inhibitory factor: evidence for a trimer 总被引:1,自引:0,他引:1
Sun Hong-Wei; Swope Melissa; Craig Cinquina; Bedarkar Sudhir; Bernhagen Jurgen; Bucala Richard; Lolis Elias 《Protein engineering, design & selection : PEDS》1996,9(8):631-635
The subunit structure of human macrophage migration inhibitoryfactor (MIF) has been studied by preliminary X-ray analysisof wild-type and selenomethionine-MIF and dynamic light scattering.Crystal form I of MIF belongs to space group P212121 and isgrown from 2 M ammonium sulfate at pH 8.5. A native data sethas been collected to 2.4 Å resolution. Self-rotationstudies and Vm values indicate that three molecules per asymmetricunit are present A data set to 2.8 Å resolution has beencollected for crystal form II, which belongs to space groupP3121 or P3221 and grows from 2 M ammonium sulfate, 2% polyethyleneglycol (average molecular mass 400), 0.1 M HEPES, pH 7.5. Three,four, five or six monomers in the asymmetric unit are consistentwith Vm values for this crystal form. Analysis of crystal formII containing selenomethionine-MIF indicates nine selenium sitesare present per asymmetric unit. Dynamic light scattering ofMIF suggests that the major form of the protein in solutionis a trimer. The results of these studies are in contrast toprevious reports indicating that MIF is a monomer or dimer.The subunit arrangement of MIF is similar to that of tumor necrosisfactor and suggests that signal transduction might require trimerizationof receptor subunits. 相似文献
4.
Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa 总被引:1,自引:0,他引:1
Medvedkin Vyacheslav N.; Permyakov Eugene A.; Klimenko Lyubov V.; Mitin Yuri V.; Matsushima Norio; Nakayama Susumu; Kretsinger Robert H. 《Protein engineering, design & selection : PEDS》1995,8(1):63-70
The proteins, AlgR3 and AlgP, are involved in the regulationof alginate synthesis in Pseudomonas. They contain multiplerepeats of Ala*Ala*Lys*Pro as do several other proteins thatresemble histones. The interactions of synthesis oligopeptidescomposed of repeated Ala*Ala*Lys*Pro or Lys*Lys*Ser*Pro unitswith DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl)group attached to the N-termini of the peptides. DNA quenchingof the Fmoc fluorescence of the peptides was used to estimatethe apparent association constants for the interaction of Fmoc(AAKP)nOH(n = 2, 4, 8, 18, 32) and of Fmoc(KKSP)nOH (n = 2, 4, 8, 16,20, 32) with DNA. The Fmoc(AAKP)nOH peptides bind to DNA onlyat low ionic strength; the Fmoc(KKSP)n OH peptides interactwith DNA at both low (0.05 M KCl) and high (0.2 M KCl) saltAt low ionic strength an increase in the number of the repeatunits causes an increase in the apparent association constantup to {small tilde}2 x 106 M1 for both types of peptidesat N 24. The insertion of an AAKTA unit into the middle ofthe Fmoc(AAKP)8OH peptide increases its affinity to DNA. Wepropose a model of (AAKP)n and of its interaction with DNA.The repeat unit consists of a single turn of -helix followedby a bend necessitated by Pro. The resultant coiled-coil formsa right-handed superhelix with 10 AAKPs per repeat distanceof {small tilde}33 Å. With only slight modification ofthe canonical parameters of this model the AAKP super helixfits into the major groove of B-form DNA with one AAKP tetramerper base pair repeat of 3.4 Å. The -amine nitrogen ofLys can form a polar hydrogen bond with a phosphate oxygen atomof the DNA backbone. A better fit is obtained when the modelis modified to accommodate [(AAKP)5AAKTA]n as actually observedin AlgR3. We suggest that this coiled-coil represents a generalmotif for other proteinDNA interactions. 相似文献
5.
Brodin Peter; Drakenberg Torbjorn; Thulin Eva; Forsen Sture; Grundstrom Thomas 《Protein engineering, design & selection : PEDS》1989,2(5):353-357
A technique for proton labelling of selected amino acids indeuterated calbindin D9K, heterologously expressed in E.coli,was developed in order to simplify and obtain higher resolutionin 1H-NMR spectra. The spectra from two double-labelling experiments,Val plus Ser and Val plus Leu, when compared to the uniformlyprotonated protein showed a dramatically simpler pattern withlow background signals and gave considerably sharper resonancesdue to reduced relaxation rates in the deuterated proteins.The selective proton labelling technique will enable detailedand rapid analysis of interesting domains of proteins and willalso make the analysis of larger proteins feasible. 相似文献
6.
Mikol Vincent; Kallen Jorg; Walkinshaw Malcolm D. 《Protein engineering, design & selection : PEDS》1994,7(5):597-603
For most of the cyclosporin A (CsA) analogs, there is generallya good correlation between cyclophilin binding and immunosuppression.However, this relationship does not seem to hold for 4-[(E)-2-butenyl]-4,4,N-trimethyl-L-threonine1(MeBm2t)1-CsA.Its affinity for cyclophilin was reported to be {small tilde}1percent; that of CsA and its immunosuppressive activity invitro was shown to be {small tilde} 30% that of CsA. We reporthere the crystal structure of a complex between recombinanthuman cyclophilin A (CypA) and (MeBm2t)1-CsA which has beendetermined by X-ray crystallography at 2.2 Å resolutionand refined to an Rfactor of 16.3%. (MeBm2t)1-CsA shows a similarbound conformation and network of interactions to CypA as CsA.The measured lower affinity for CypA cannot therefore be explainedby a different mode of binding. We propose that the poor affinityto CypA could be accounted for by the existence of an equilibriumin aqueous solution between a cyclophilin bound conformationand a nonbinding conformation of (MeBm2t)1-CsA.The relatively high immunosuppressive activity is suggestedto result from slight conformational differences observed inthe effector domain 相似文献
7.
Corbier C.; Branlant C.; Wonacott A.; Branlant G. 《Protein engineering, design & selection : PEDS》1989,2(7):559-562
A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphatedehydrogenase, Ser148 Ala, was produced byoligonucleotide-directedmutagenesis. The study of the catalytic properties of this mutanthas shown that this mutation significantly affects the Michaelisconstant of inorganic phosphate and to a lesser extent thatof 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate.This result is consistent with model-building studies whichshow that, for the phosphorylation step of catalysis, inorganicphosphate must bind to the anion recognition site designatedPi with the C(3) phosphate of the acyl-enzyme intermediate inthe alternative anion site Ps. Studies of the enantiomeric specificityusing D- and L-glyceraldehyde as substrates show that the hydroxylgroup of Ser148, combined with the presence of the C(3) phosphateof the substrate, enhances stereospecificity as well as catalysis.However, the stereospecific effect cannot be a consequence ofthe direct interaction of Ser148 with the C(2)-hydroxyl of thesubstrate. The changed Km for glyceraldehyde-3-phosphate suggeststhat the initial step of hemithioacetal formation may take placewith its C(3) phosphate bound in the Pi site. This supportsthe molecular mechanism proposed by Moody (1984). Therefore,catalysis could be enhanced through interactions of the serinehydroxyl group not only with inorganic phosphate but also withthe C(3) phosphate of glyceraldehyde-3-phosphate. 相似文献
8.
Ueda Tadashi; Yamada Hidenori; Imoto Taiji 《Protein engineering, design & selection : PEDS》1987,1(3):189-193
In the cross-linking reaction of lysozyme between Leu129 (-COO)and Lys13 (-NH3+ using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimidehydrochloride (EDC), a side reaction of the peptide bond inversionfrom to ß between A and Gly102 was greatly reducedby addition of ß-(1,4)-linked trimer of N-acetyl-D-glucosamine[(NAG)3] When methylamine or 2-hydroxyethylamine was furtheradded, the extent of the cross-link formation was decreasedand the derivative where the -carboxyl group of Leu129 was modifiedwith the amine was newly obtained. On the other hand, when ammoniawas added, the ß-carboxyl group of Asp119 insteadof the -carboxyl group was mainly amidated. From these results,the presence of a salt bridge between Asp119 and Arg125 besidesthat between Lys13 and Leu129 is proposed. Enzymatic activitiesof the derivatives prepared here indicated that the modificationof the -carboxyl group reduced the activity to {small tilde}90% of that of native lysozyme. Des-Leu129 lysozyme, which lacksLeu129 also showed {small tilde} 90% of the activity of nativelysozyme. Therefore, the salt bridge between Lys13 and Leu129may play some role in maintaining the active conformation oflysozyine. 相似文献
9.
Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6 总被引:1,自引:0,他引:1
Jean Lee-Fong L.; Waters Cory A.; Keemy Donald; Murphy John R. 《Protein engineering, design & selection : PEDS》1993,6(3):305-311
We have used site-directed and in-frame deletion mutationalanalysis in order to explore the structural features of theIL6 portion of the diphtheria toxin-related interleukin6(IL6) fusion toxin DAB389-IL6 that are essentialfor receptorbinding and subsequent inhibition of protein synthesisin target cells. Deletion of the first 14 amino acids of theIL6 component of the fusion toxin did not alter eitherreceptor binding affinity or cytotoxk potency. In contrast,both receptor binding and cytotoxic activity were abolishedwhen the Cterminal 30 amino acids of the fusion toxinwere deleted. In addition, we explored the relative role ofthe disulfide bridges within the IL6 portion of DAB389-IL6in the stabilization of structure required for receptor-binding.The analysis of mutants in which the substitution of eitherCys440, Cys446, Cys469 or Cys479 to Ser respectively, demonstratesthat only the disulfide bridge between Cys469 and Cys479 isrequired to maintain a functional receptor binding domain. Inaddition, the internal in-frame deletion of residues 435451,which includes Cys440 and Cys446, was found to reduce, but notabolish receptor binding affinity. These results further demonstratethat the disulfide bridge between Cys440 and Cys446 is not essentialfor receptor-binding. However, the reduced cytotoxic potencyof DAB389-IL6(435451) suggests that the conformationand/or receptor binding sites associated with this region ofthe fusion toxin is/are important for maintaining the wild typereceptor binding affinity and cytotoxic potency. 相似文献
10.
Dealwis Chris G.; Chen Liqing; Brennan Catherine; Mandecki Wlodek; Abad-Zapatero Cele 《Protein engineering, design & selection : PEDS》1995,8(9):865-871
The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (kcat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased kcat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg2+binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg2+, and the loop Glnl52Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg2+ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg2+ ion, and alsoby the loss of an electrostatic interaction (Mg2+.COO-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg2+ ion, arefurther apart in the mutant compared with the wild-type 相似文献
11.
Hakoshima Toshio; Toda Shoji; Sugio Shigetoshi; Tomita Ken-ichi; Nishikawa Satoshi; Morioka Hiroshi; Fuchimura Kayoko; Kimura Tsuyoshi; Uesugi Sei-ichi; Ohtsuka Eiko; Ikehara Morio 《Protein engineering, design & selection : PEDS》1988,2(1):55-61
Recognition by ribonuclease T1 of guanine bases via multidentatehydrogen bonding and stacking interactions appears to be mediatedmainly by a short peptide segment formed by one stretch of aheptapeptide, Tyr42-Asn43-Asn44-Tyr45-Gly46-Gly47-Phe48. Thesegment displays a unique folding of the polypeptide chainconsistingof a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by ahydrogen-bond network involving the side chain of Asn44, themain-chain atoms of Asn44, Gly47 and Phe48 and one water molecule.The segment is connected to the C terminus of a ß-strandand expands into a loop region between Asn43 and Ser54. Lowvalues for the crystallographic thermal parameters of the segmentindicate that the structure has a rigidity comparable to thatof a ß-pleated sheet. Replacement of Asn44 with alanineleads to a far lower enzymatic activity and demonstrates thatthe side chain of Asn44 plays a key role in polypeptide foldingin addition to a role in maintaining the segment structure.Substitution of Asn43 by alanine to remove a weak hydrogen bondto the guanine base destabilized the transition state of thecomplex by 6.3 kJ/mol at 37°C. In contrast, mutation ofGlu46 to alanine to remove a strong hydrogen bond to the guaninebase caused a destabilization of the complex by 14.0 kJ/mol.A double-mutant enzyme with substitutions of Asn43 by a histidineand Asn44 by an aspartic acid, to reproduce the natural substitutionsfound in ribonuclease Ms, showed an activity and base specificitysimilar to that of the wild-type ribonuclease Ms. The segmenttherefore appears to be well conserved in several fungal ribonucleases. 相似文献
12.
The influenza A M2 protein forms cation-selective ion channelswhich are blocked by the anti-influenza drug amantadine. A molecularmodel of the M2 channel is presented in which a bundle of fourparallel M2 transbilayer helices surrounds a central ion-permeablepore. Analysis of helix amphipathicity was used to aid determinationof the orientation of the helices about their long axes. Thehelices are tilted such that the N-terminal mouth of the poreis wider than the C-terminal mouth. The channel is lined byresidues V27, S31 and I42. Residues D24 and D44 are locatedat opposite mouths of the pore, which is narrowest in the vicinityof I42. Energy profiles for interaction of the channel withNa+, amantadine-H+ and cyclopentylamine-H+ are evaluated. Theinteraction profile for Na+ exhibits three minima, one at eachmouth of the pore, and one in the region of residue S31. Theamantadine-H+ profile exhibits a minimum close to S31 and abarrier near residue I42. This provides a molecular model foramantadine-H+ block of M2 channels. The profile for cyclopentylamine-H+does not exhibit such a barrier. It is predicted that cyclopentyl-amine-H+will not act as an M2 channel blocker. 相似文献
13.
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
14.
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
15.
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
16.
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
17.
Altering the association properties of insulin by amino acid replacement 总被引:12,自引:0,他引:12
Brems David N.; Alter Leila A.; Beckage Michael J.; Chance Ronald E.; DiMarchi Richard D.; Green L. Kenney; Long Harlan B.; Pekar Allen H.; Shields James E.; Frank Bruce H. 《Protein engineering, design & selection : PEDS》1992,5(6):527-533
The importance of ProB28 and LysB29 on the self-associationof insulin was established by systematically truncating theC terminus of the B chain. The relationship between structureand association was further explored by making numerous aminoacid replacements at B28 and B29 Association was studied bycircular dichroism, size-exclusion chromatography and ultracentrifugation.Our results show that the location of a prolyl residue at B28is critical for high-affinity self-association. Removal of ProB28in a series of C-terminal truncated insulins, or amino acidreplacement of Pro28, greatly reduced association. The largestdisruption to association was achieved by replacing LysB29 withPro and varying the amino acid at B28 Several of the analogswere predominantly monomers in solutions up to 3 mg/ml. Theseamino acid substitutions decreased association by primarilydisrupting the formation of dimers. Such amino acid substitutionsalso substantially reduced the Zn-induced insulin hexamer formation.The formation of monomeric insulins through amino acid replacementswas accompanied by conformational changes that may be the causefor decreased association. It is demonstrated that self-associationof insulin can be drastically altered by substitution of oneor two key amino acids. 相似文献
18.
Vallera Daniel A.; Seo Su-Yeong; Panoskaltsis-Mortari Angela; Griffin James D.; Blazar Bruce R. 《Protein engineering, design & selection : PEDS》1999,12(9):779-785
The IL-3 receptor was expressed on a high frequency of myeloidleukemia cells and also on hematopoietic and vascular cells.We previously showed that a recombinant IL-3 fusion immunotoxin(DT390IL-3) expressed by splicing the murine IL-3 gene to atruncated diphtheria toxin (DT390) gene selectively killed IL-3R+expressing cells and was not uniformly toxic to uncommited BMprogenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J.and Vallera,D.A., 1996, Blood, 88, 14451456). Thus, weexplored the feasability of using DT390IL-3 as an anti-leukemiaagent. DT390IL-3 was toxic when administered to mice at dosesas low as 0.1 µg/day. The dose limiting toxicity appearedto be related to platelet and bleeding effects of the fusiontoxin. Because of these effects, DT390IL-3 was studied ex vivoas a means of purging contaminating leukemia cells from BM graftsin a murine autologous BM transplantation. In this setting,as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid32Dp210 leukemia cells were lethal. An optimal purging intervalof 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BMmixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneicmice. Mice given treated grafts containing BM and a lethal doseof 32Dp210 cells survived over 100 days while mice given untreatedgrafts did not survive (P < 0.00001). DT390IL-3 may provehighly useful for ex vivo purging of lethal malignant leukemiacells from autologous BM grafts. 相似文献
19.
Adib-Conquy Minou; Gilbert Michele; Christodoulou Christina; Avrameas Stratis 《Protein engineering, design & selection : PEDS》1995,8(9):859-863
In this report, we describe the expression system that enabledus to produce in Escherichia coli the Fab fragment of a mouseIgM that has previously been shown to inhibit the binding ofIgG to autoantigens by interacting with their variable regions.In our system, both light chain and heavy chain fragments wereput under the control of the malE promoter. The light chainwas fused to the MalE signal sequence, while the heavy chainvariable and first constant region were fused to the alkalinephosphatase signal sequence. In this system, after inductionof the promoter with maltose, the Fab fragment could be detectedin a periplasmic extract of the bacteria by Western blottingand also by ELISA. This Fab fragment was purified on a goatanti-mouse immunoglobulin immunoadsorbent and biotinylated.The Fab fragment produced by E.coli reacted with the trinitrophenyl(TNP) hapten and F(ab')2 fragments of mouse IgG and these reactivitiescould be specifically inhibited by the corresponding solubleantigens. The dissociation constants of this Fab were 1.65 x106 M for TNP and 5 x 106 M for IgG F(ab')2 fragments,indicating that the affinity of the Fab fragment compared withthat of the whole IgM molecule was similar for TNP but was lowerfor IgG F(ab')2 fragments 相似文献
20.
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