共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
Kusakabe Takahiro; Motoki Kiyohisa; Sugimoto Yasushi; Takasaki Yozo; Hori Katsuji 《Protein engineering, design & selection : PEDS》1994,7(11):1387-1393
A series of chimeric enzymes between two human aldolases A,B or C were constructed to identify the molecular regions responsiblefor isozyme-specific functions. Chimeras constructed betweenaldolases A and B were AB34 (an AB chimera connected at position34), ABA34306 and ABA212306 (the ABA chimeras).Those between aldolases B and C are BC243, BC263 and BC306 (theBC chimeras connected at positions as indicated), as well asCB55, CB243, CB263 and CB306 (the CB chimeras connected at positionsas indicated), CBC55263 (a CBC chimera), and BCB55193,BCB55306, BCB79193 and BCB79306 (the BCBchimeric enzymes). Through the analysis of the properties ofthese chimeras, it was found that for aldolase B, isozyme Bgroup-specific sequences (IGSs)-l and-4 were required for exertingtype B-specific functions, while the IGSs-2 and -3 enhanced,in collaboration with the IGS-1, the catalytic activity of aldolaseB. In addition, the /ß-barrel and the restricted stretches,which were not specified but occupied two distinct regions spanningthe amino acid positions 108137 (designated connector1) and 243306 (designated connector 2), were found tobe indispensable for showing full catalytic activity of aldolaseB. 相似文献
3.
Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat 总被引:1,自引:0,他引:1
Mandecki Wlodek; Shallcross Mary Ann; Sowadski Janusz; Tomazic-Allen Susan 《Protein engineering, design & selection : PEDS》1991,4(7):801-804
The likelihood for improvement in the catalytic properties ofEscherichia coli alkaline phosphatase was examined using site-directedmutagenesis. Mutants were constructed by introducing sequencechanges into nine preselected amino acid sites within 10 A ofthe catalytic residue serine 102. When highly conserved residuesin the family of alkaline phosphatases were mutated, many ofthe resulting enzymes not only maintained activity, but alsoexhibited greatly improved tra,. Of 170 mutant enzymesscreened, 5% (eight mutants) exhibited significant increasesin specific activity. In particular, a substitution by serineof a totally invariant AsplOl resulted in a 35-fold increaseof specific activity over wild-type at pH 10.0. Up to 6-foldincreases the kcat/km ratio were observed. 相似文献
4.
Copeland William C.; Vanderslice Peter; Robertus Jon D. 《Protein engineering, design & selection : PEDS》1987,1(5):419-423
The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the Km of wild-type enzymeis 0.4 mM and the kcat = 2800 min1; the correspondingvalues for G58D are 0.5 mM and 2750 min1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K+ and pH 7.0.At pH 7.6 and 0.8 M K+ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate. 相似文献
5.
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.
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
8.
Simons Guus; van den Heuvel Wim; Reynen Theo; Frijters Adri; Rutten Ger; Slangen Charles J.; Groenen Martien; de Vos Willem M.; Siezen Roland J. 《Protein engineering, design & selection : PEDS》1993,6(7):763-770
A cDNA clone containing the entire coding region for bovineß-casein A3 flanked by 53 base pairs of 5' non-codingand 358 base pairs of 3' non-coding sequences was isolated froma bovine mammary cDNA phagemid library. The coding segment formature ß-casein was subcloned into the T7 expressionsystem, in which the expression of recombinant ß-caseinwas controlled by the T7 gene 10 promoter and ribosome bindingsite. High level expression of Met-ß-casein to 20%of the total soluble proteins was obtained in Escherichia coliwithin 2 h after induction of T7 RNA-polymerase synthesis. Inan attempt to induce secretion the coding segment for matureß-casein was coupled to the ompA translations initiationsignal and signal peptide coding sequence but no secretion ofthe fusion protein and no processing of the signal peptide fromthe fusion protein was observed. Instead, the Met-ß-caseincould be isolated in asoluble form from E.coli cells after anosmotic shock, indicative of a periplasmic location. This proceduredid not lyse the cells. The protein was purified to homogeneityafter a pH 4.8 isoelectric precipitation followed by reversed-phasehigh-performance liquid chromatography. The ß-caseincDNA was altered to change the main chymosin cleavage siteinß-casein at position 192193 in two ways, namelyfrom LeuTyr to ProPro and to Leustop. Thesemutations were designed to prevent generation of the bitterpeptide ßcasein(193209) by chymosin cleavage.The mutant Met-ß-caseins were expressed in E.colito the same level as wild-type Met-ß-casein. Purifiedmutant Met-ß-casein(Prol92 Prol93) was no longerhydrolysed by chymosin at the 192193 bond. 相似文献
9.
Structure and functional complementation of engineered fragments from yeast phosphoglycerate kinase 总被引:1,自引:0,他引:1
Pecorari Frederic; Minard Philippe; Desmadril Michel; Yon Jeannine M. 《Protein engineering, design & selection : PEDS》1993,6(3):313-324
Previous studies have shown that, although the isolated structuraldomains of yeast phosphoglycerate kinase recover a quasi-nativestructure in vitro as well as in vivo, they do not reassociatenor generate a functional enzyme. The aim of this work was firstto study the folding of complementary fragments different fromstructural domains and second to determine the requirementsfor their reassociation and functional complementation. Themethod used for producing rigorously defined fragments consistsof the introduction of a unique cysteinyl residue in the proteinfollowed by a specific cleavage by 5'5'-dithiobis(2-nitrobenzoate)/potassiumcyanide at this residue. Two pairs of complementary fragmentswere thus obtained, 196/97415 and 1248/249415.The structure and stabilities of the different fragments werestudied. The short fragments, i.e. 196 and 249415were found to contain some secondary structure, but to havea low stability. Each large fragment has a high structural contentand a stability close to that of the corresponding domain. Incontrast to that observed with the isolated domains, a weakbut significant complementation was observed for the two pairsof fragments; the pair of fragments 1248/249415recovered 8% of the activity of the native enzyme upon complementation.An independent refolding of the complementary fragments beforereassociation decreased the yield of complementation for thepair of fragments 196/97415, but did not affectthe complementation for the other pair (1248/249415).From the present data and previous work on the isolated domains,it appears that the correct folding of the isolated fragmentsis not a prerequisite for their complementation. 相似文献
10.
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
11.
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
12.
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
13.
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
14.
Eidsness Marly K.; O'Dell Sonia E.; Kurtz Donald M. Jr; Robson Robert L.; Scott Robert A. 《Protein engineering, design & selection : PEDS》1992,5(4):367-371
A synthetic gene based on the published amino acid sequencefor Clostridium pasteurianum rubredoxin was constructed, clonedin Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promotersystem in E.coli HMS273. UV/visible spectroseopy and metal analysesindicated that the asisolated synthetic gene productis a mixture of holo(i.e. ironcontaining) rubredoxinand zincsubstituted rubredoxin, with the latter amountingto {small tilde} 70% of the total rubredoxin. Hie UV/visibleabsorption and resonance Raman spectra of the cloned holorubredoxinare characteristic of the native rubredoxintype ironsite. Nterminal amino acid sequencing suggests that thegene product consists of at least three polypeptide specieswith the initial sequences (approximate relative abundances):MetMetLys... (63%), blocked (30%) and MetLys...(7%). The blocked portion presumably consists of a mixture ofnMetMetLys... and nMet-Lys..., wherenMet represents an aminoblocked methionine residue. 相似文献
15.
Nishikawa Satoshi; Adiwinata Jeanne; Morioka Hiroshi; Fujimura Takao; Tanaka Toshiki; Uesugi Seiichi; Hakoshima Toshio; Tomita Ken-ichi; Nakagawa Setsuko; Ikehara Morio 《Protein engineering, design & selection : PEDS》1990,3(5):443-448
Molecular-dynamic calculations predict that, if Tyr24 and Asn84are each replaced by a Cys residue, it should be possible toform a third disulfide bond in ribonuclease T1 (RNase T1) betweenthese residues, with only minimal conformational changes atthe catalytic site. The gene encoding such a mutant variantof RNase T1 (Tyr24 Cys24, Asn84 Cys84) was constructedby the cassette mutagenesis method using a chemically synthesizedgene. In order to reduce the toxic effect of the mutant enzyme(RNase T1S) on an Escherichia coli host, we arranged for theprotein to be secreted into the periplasmic space by using avector that harbors a gene for an alkaline phosphatase signalpeptide under the control of the trp promoter. The nucleolyticactivity of RNase T1S toward pGpC was approximately the sameas that of RNase T1 at 37°C (pH 7.5). Moreover, at 55°C,RNase T1S retained nearly 70% of its activity while the activityof the wild-type enzyme was reduced to <10%. RNase T1S wasalso more resistant to denaturation by urea than the wild-typeenzyme. However, unlike RNase T1, RNase T1S was irreversiblyand almost totally inactivated by boiling at 100°C for 15min. 相似文献
16.
Ducancel FrEdEric; Boulain Jean-Claude; TrEmeau Odile; MEnez AndrE 《Protein engineering, design & selection : PEDS》1989,3(2):139-143
We constructed a recombinant expression plasmid encoding a proteinAneurotoxin fusion protein. The fused toxin is directlyexpressed in the periplasmic space of Escherichia coli and canbe purified in the milligram range by a single immuno-affinitystep. The LD50 values of the fused toxin and native toxin are130 and 20 nmol/kg mouse respectively. The Kd values characterizingtheir binding to the nicotinic acetylcholine receptor (AcChoR)are respectively 4.8 ± 0.8 and 0.07 ± 0.03 nM.In contrast, the fused and native toxins are equally well recognizedby a toxin-specific monoclonal antibody which recognizes theAcChoR binding site. The lower toxicity of the fused toxin mightresult, therefore, from a steric hindrance, due to the presenceof the bulky protein A moiety (mol. wt = 31 kd) rather thanto a direct alteration of the toxic site. Thefused toxin is more immunogenic than native toxin, since 1 nmolof hybrid toxin and 14 nmol of native toxin give rise to comparabletiters of antitoxin antibodies which, furthermore, are equallypotent at neutralizing neurotoxicity. The work described inthis paper shows that the use of fused toxins may be of paramountimportance for future development of serotherapy against envenomationby snake bites. 相似文献
17.
Dolgikh Dmitry A.; Uversky Vladimir N.; Gabrielian Andrey E.; Chemeris Violetta V.; Fedorov Alexey N.; Navolotskaya Elena V.; Zav'yalov Vladimir P.; Kirpichnikov Michael P. 《Protein engineering, design & selection : PEDS》1996,9(2):195-201
The de novo protein albebetin has been designed recently toform a predetermined tertiary fold that has not yet been observedin natural proteins. An eight amino acid fragment (131138)of human interferon 2 carrying the blasttransforming activityof the protein was attached to the N-terminus of albebetin nextto its initiatory methionine residue. The gene of chimeric proteinwas expressed in a wheat germ cell-free translation system andsynthesized protein was tested for its compactness and stability.Its ability for receptor binding was also studied. We have shownthat albebetin with attached octapeptide is practically as compactas natural proteins of corresponding molecular weight and possesseshigh stability toward the urea-induced unfolding. It binds murinethymocyte receptor at a high affinity and activates the thymocyteblast transformation efficiently at a concentration of 10-11M. 相似文献
18.
Gore Michael G.; Ferris William F.; Popplewell Andrew G.; Scawen Michael; Atkinson Tony 《Protein engineering, design & selection : PEDS》1992,5(6):577-582
A fusion protein, consisting of the N-terminal 81 amino acidsfrom an inactive bovine DNase I (Q38,E39E38,Q39) andtwo sequential synthetic IgG-binding domains based upon domainB of Protein A from Staphylococcus aureus has been shown tobind to porcine IgG with a similar affinity and pH profile toProtein A. The same residue in each B domain (Tyr111 and Tyr169)has been mutated by cassette mutagenesis to Ser, Glu, His, Lysor Arg and the effect of the mutation on binding interactionswith porcine IgG investigated. The evidence presented suggeststhat the interactions at the B domain are highly sensitive tothe presence of a charged residue. 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.
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