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1.
Harford-Cross Charles F.; Carmichael Angus B.; Allan Fiona K.; England Paul A.; Rouch Duncan A.; Wong Luet-Lok 《Protein engineering, design & selection : PEDS》2000,13(2):121-128
Mutations of the active site residues F87 and Y96 greatly enhancedthe activity of cytochrome P450cam (CYP101) from Pseudomonasputida for the oxidation of the polycyclic aromatic hydrocarbonsphenanthrene, fluoranthene, pyrene and benzo[a]pyrene. Wild-typeP450cam had low (<0.01 min1) activity with these substrates.Phenanthrene was oxidized to 1-, 2-, 3- and 4-phenanthrol, whilefluoranthene gave mainly 3-fluoranthol. Pyrene was oxidizedto 1-pyrenol and then to 1,6- and 1,8-pyrenequinone, with smallamounts of 2-pyrenol also formed with the Y96A mutant. Benzo[a]pyrenegave 3-hydroxybenzo[a]pyrene as the major product. The NADHoxidation rate of the mutants with phenanthrene was as highas 374 min1, which was 31% of the camphor oxidation rateby wild-type P450cam, and with fluoranthene the fastest ratewas 144 min1. The oxidation of phenanthrene and fluoranthenewere highly uncoupled, with highest couplings of 1.3 and 3.1%,respectively. The highest coupling efficiency for pyrene oxidationwas a reasonable 23%, but the NADH turnover rate was slow. Theproduct distributions varied significantly between mutants,suggesting that substrate binding orientations can be manipulatedby protein engineering, and that genetic variants of P450cammay be useful for studying the oxidation of polycyclic aromatichydrocarbons by P450 enzymes. 相似文献
2.
We had reported engineering of the heme monooxygenase cytochrome P450cam from Pseudomonas putida with the F87W/Y96F/L244A/V247L mutations for the oxidation of pentachlorobenzene (PeCB), a recalcitrant environmental contaminant, to pentachlorophenol. In order to provide further insights into P450 structure, function and substrate recognition, we have determined the crystal structure of this 4-mutant without a substrate and its complex with PeCB. PeCB is bound face-on to the heme, with a weak Fe--Cl interaction. One PeCB chlorine is located in the cavity generated by the L244A mutation, in striking illustration of the role of this mutation in promoting PeCB binding. The structures also show that the P450(cam) oxygen-binding groove between G248 and T252 is flexible and can tolerate significant deviations from their conformations in the wild type without loss of enzyme activity. Analysis of the PeCB binding interactions led to introduction of the T101A mutation to enable the substrate to reorient during the catalytic cycle for more efficient oxidation. The resultant 5-mutant F87W/Y96F/T101A/L244A/V247L is 3-fold more active for PeCB oxidation than the 4-mutant. Polychlorinated benzene binding by the mutants and the partitioning between substrate oxidation and non-productive (uncoupling) side reactions are correlated with the structural data. 相似文献
3.
Thiocamphor is an unusual substrate for P450cam in that in theX-ray structure it binds in the active site pocket in two distinctorientations and neither of these orientations are consistentwith the 5-alcohol being the primary product. Other camphoranalogs such as norcamphor or camphane bind in a single orientationconsistent with the 5-alcohol being a major product. We presentan analysis of four 175 ps molecular dynamics trajectories ofthiocamphor-bound cytochrome P450cam. The first two trajectorieswere calculated for cytochrome P450cam with thiocamphor boundin both its major and minor crystallographic orientations. Inthe second set of simulations, a single oxygen atom was addedas a distal ligand to the heme group in order to model the putativeferryl oxygen reaction intermediate. Trajectories were againcalculated starting with thiocamphor in its major and minororientations. While the protein dynamics were quite similarin all four trajectories, the substrate showed distinctly differentmotions in each of the trajectories. In particular, the preferredsubstrate orientations were very different in the presence ofthe ferryl oxygen than in the absence of that oxygen. The preferredorientations in the absence of the distal oxygen were consistentwith the 3-akohol being the major product, while the preferredorientations in the presence of the distal oxygen were consistentwith the 5-alcohol being a major product. These simulationsoffer an explanation for the inconsistency between the X-raydata and the product profile. 相似文献
4.
Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity 总被引:1,自引:0,他引:1
Zapata Gerardo; Ridgway John B. B.; Mordenti Joyce; Osaka Gary; Wong Wai Lee T.; Bennett Gregory L.; Carter Paul 《Protein engineering, design & selection : PEDS》1995,8(10):1057-1062
We developed a novel bivalent antibody fragment, the linear(L-) F(ab')2, comprising tandem repeats of a heavy chain fragmentVHCH1VHCH1 cosecreted with a light chain.Functional humanized L-F(ab')2 directed against p185HER2 wassecreted from Escherichia coli at high titer (100 mg/l) andpurified to homogeneity. The L-F(ab')2 binds two equivalentsof antigen with an apparent affinity (Kd = 0.46 nM) that iswithin 3-fold of the corresponding thioether-linked F(ab')2fragment The N-terminal site binds antigen with an affinity(Kd = 1.2 nM) that is 4-fold greater than that for the C-terminalsite, as shown by the comparison of L-F(ab')2 variants containinga single functional binding site. L-F(ab')2 has greater antiproliferativeactivity than the thioether-linked F(ab')2 against the p185HER2-overexpressingtumor cell line BT474. Linear and thioether-linked F(ab')2 havevery similar pharmacokinetic properties in normal mice, andtheir serum permanence times are respectively 7- and 8-foldlonger than the corresponding Fab fragment L-F(ab')2 offersa facile route to bivalent antibody fragments that are potentiallysuitable for clinical applications, and that may have improvedbiological activity compared with thioether-linked F(ab')2 fragments. 相似文献
5.
Thunnissen M.G.M. Marjolein; Franken Peet A.; de Haas Gerard H.; Drenth Jan; Kalk Kor H.; Verheij Hubertus M.; Dijkstra W. 《Protein engineering, design & selection : PEDS》1992,5(7):597-603
Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A2. They are part of an extendedhydrogen bonding system that links the N-terminal -NH+3 -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA2 mutant, in which residues 6266 had been deleted (6266PLA2).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 6266PLA2, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization. 相似文献
6.
Kuipers Oscar P.; Kerver Jana; van Meersbergen Joop; Vis Roel; Dijkman Ruud; Verheij Hubertus M.; de Haas Gerard H. 《Protein engineering, design & selection : PEDS》1990,3(7):599-603
Residue 31 of porcine pancreatic phospholipase A2 (PLA2) islocated at the entrance to the active site. To study the roleof residue 31 in PLA2, six mutant enzymes were produced by site-directedmutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Seror Gly. Direct binding studies indicated a three to six timesgreater affinity of the Trp31 PLA2 for both monomeric and micellarsubstrate analogs, relative to the wild-type enzyme. The otherfive mutants possess an unchanged affinity for monomers of theproduct analog n-decylphosphocholine and for micelles of thediacyl substrate analog rac-l,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine.The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholinewere decreased 920 times for these five mutants. Kineticstudies with monomeric substrates showed that the mutants haveVmax values which range between 15 and 70% relative to the wild-typeenzyme. The Vmax values for micelles of the zwitterionic substratel,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 350times. The Km values for the monomeric substrate and the kmvalues for the micellar substrate were hardly affected in thecase of five of the six mutants, but were considerably decreasedwhen Trp was present at position 31. The results of these investigationspoint to a versatile role for the residue at position 31: involvementin the binding and orientating of monomeric substrate (analogs),involvement in the binding of the enzyme to micellar substrateanalogs and possibly involvement in shielding the active sitefrom excess water. 相似文献
7.
Nandagopal Krishnadas; Tadaki Douglas K.; Lamerdin John A.; Serpersu Engin H.; Niyogi Salil K. 《Protein engineering, design & selection : PEDS》1996,9(9):781-788
The biological importance of Leu15 of epidermal growth factor(EGF) is suggested by its conservation through evolution, itscritical location in the domaindomain interface of EGFand its close proximity to Arg41, a residue that is crucialfor receptor binding and activation. Mutagenesis of Leu15 ofhuman EGF (hEGF) was employed to examine the role of this residuein the ligand-receptor interaction. The relative receptor affinitiesof the hEGF variants, as determined by radioreceptor competitionassays, varied depending on the amino acid substitution. TheL15F, L15W and L15V hEGF analogues had receptor affinities 45,26 and 18% respectively of wild type hEGF. The L15A and L15Ranalogues displayed receptor affinities of only 2.4 and 1.6%relative to wild type hEGF. No binding of the L15E analoguewas detected. The relative agonist activities, as measured byreceptor tyrosine kinase stimulation assays, generally followeda similar trend. The L15F, L15W and L15V analogues stimulatedthe receptor kinase to a level (Vmax) similar to that for wildtype hEGF. A striking difference was observed between the L15Aand L15R variants; although having similar binding affinities,the L15A mutant activated the receptor to only {small tilde}5%of the wild type Vmax in contrast to 53% for the L15R mutant1H-NMR analysis of the L15R and L15A mutants showed only minorstructural alterations that were not sufficient to account forthe dramatic losses in binding and agonist activities. The resultsindicate that both the size and hydrophobicity of the -branchedaliphatic side chain of Leu15 of hEGF are important in the formationof a catalytically active ligandreceptor complex. 相似文献
8.
Turner S.L.; Ford G.C.; Mountain A.; Moir A. 《Protein engineering, design & selection : PEDS》1992,5(6):535-541
The moderate thermophile Bacillus stearothermophilus was usedas a host in which to detect more thermostable variants of theB.pumilus chloramphenicol acetyltransferase (Cat-86) protein.Seventeen mutants were isolated and detected by their abilityto grow in the presence of chloramphenicol at a previously restrictivetemperature (58°C). The genes encoding these proteins weresequenced; all 17 mutants carried the same C to T transitionthat conferred an amino acid substitution of alanine by valineat position 203 of the protein sequence. The wild-type and onemutant Cat-86 protein were purified to homogeneity using affinitychromatography, and kinetic and thermal stability studies wereundertaken. Both enzymes had similar sp. act. in the regionof 215 U/mg, with Km values for chloramphenicol in the range13.815.4 µM and for acetyl CoA in the range 13.615.5µM. The A203V mutant shows greater stability than thewild-type Cat-86 protein at temperatures above 50°C andappears to pass through a transition state between 48 and 50°C. 相似文献
9.
Kojima Shuichi; Miyoshi Katsunori; Miura kin-ichiro 《Protein engineering, design & selection : PEDS》1996,9(12):1241-1246
Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of thesquash-type protease inhibitor family, is composed of 29 aminoacids and shows strong inhibition of trypsin by its compactstructure. To study the structurefunction relationshipof this inhibitor using protein engineering methods, we constructedan expression system for CMTI-I as a fused protein with porcineadenylate kinase (ADK). A Met residue was introduced into thejunction of ADK and CMTI-I to cleave the fusion protein withCNBr, whereas a Met at position 8 of authentic CMTI-I was replacedby Leu. Escherichia coli JM109 transformed with the constructedplasmid expressed the fused protein as an inclusion body. Aftercleavage of the expressed protein with CNBr, fully reduced speciesof CMTI-I were purified by reversed-phase HPLC and then oxidizedwith air by shaking. For efficient refolding of CMTI-I, we used50 mM NH4HCO3 (pH 7.8) containing 0.1% PEG 6000 at higher proteinconcentration. Strong inhibitory activity toward trypsin wasdetected only in the first of three HPLC peaks. The inhibitorconstant of CMTI-I thus obtained, in which Met8 was replacedby Leu, was 1.4x10-10 M. The effect of replacement of Met withLeu at position 8 was shown to be small by comparison of theinhibitor constant of authentic CMTI-III bearing Lys at position9 (8.9x10-11 M) with that of its mutant bearing Leu at position8 and Lys at position 9 (1.8x10-10 M). To investigate the roleof the well conserved hydrophobic residues of CMTI-I in itsinteraction with trypsin, CMTI-I mutants in which one or allof the four hydrophobic residues were replaced by Ala were prepared.The inhibitor constants of these mutants indicated that thosewith single replacements were 540 times less effectiveas trypsin inhibitors and that the quadruple mutant was 450times less effective, suggesting that the hydrophobic residuesin CMTI-I contribute to its tight binding with trypsin. However,each mutant was not converted to a temporary inhibitor. 相似文献
10.
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
11.
Peelman Frank; Vanloo Berlinda; Perez-Mendez Oscar; Decout Anne; Verschelde Jean-Luc; Labeur Christine; Vinaimont Nicole; Verhee Annick; Duverger Nicolas; Brasseur Robert; Vandekerckhove Joel; Tavernier Jan; Rosseneu Maryvonne 《Protein engineering, design & selection : PEDS》1999,12(1):71-78
Lecithin cholesterol acyltransferase (LCAT) is an interfacialenzyme active on both high-density (HDL) and low-density lipoproteins(LDL). Threading alignments of LCAT with lipases suggest thatresidues 5074 form an interfacial recognition site andthis hypothesis was tested by site-directed mutagenesis. The(5668) deletion mutant had no activity on any substrate.Substitution of W61 with F, Y, L or G suggested that an aromaticresidue is required for full enzymatic activity. The activityof the W61F and W61Y mutants was retained on HDL but decreasedon LDL, possibly owing to impaired accessibility to the LDLlipid substrate. The decreased activity of the single R52A andK53A mutants on HDL and LDL and the severer effect of the doublemutation suggested that these conserved residues contributeto the folding of the LCAT lid. The membrane-destabilizing propertiesof the LCAT 5668 helical segment were demonstrated usingthe corresponding synthetic peptide. An M65NN66M substitutiondecreased both the fusogenic properties of the peptide and theactivity of the mutant enzyme on all substrates. These resultssuggest that the putative interfacial recognition domain ofLCAT plays an important role in regulating the interaction ofthe enzyme with its organized lipoprotein substrates. 相似文献
12.
McCartney John E.; Tai Mei-Sheng; Hudziak Robert M.; Adams Gregory P.; Weiner Louis M.; Jin Donald; Stafford Walter F. III; Liu Sen; Bookman Michael A.; Laminet Axel A.; Fand Irwin; Houston L. L.; Oppermann Hermann; Huston James S. 《Protein engineering, design & selection : PEDS》1995,8(3):301-314
Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv')have been engineered using model sFv proteins based upon the26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonalantibodies. As part of the 741F8 sFv construction process, thePCR-amplified 741F8 VH gene was modified in an effort to correctpossible primer-induced errors. Genetic replacement of the N-terminalp-strandsequence of 741F8 VH with that from the FR1 of anti-c-erbB-2520C9 VH resulted in a dramatic improvement of sFv folding yields.Folding in urea-glutathione redox buffers produced active sFv'with a protected C-terminal sulfhydryl, presumably as the mixeddisulfide with glutathione. Disulfide-bonded (sFv')2 homodimerswere made by disulfide interchange or oxidation after reductiveelimination of the blocking group. Both 2610 (sFv')2and 741F8 (sFv')2 existed as stable dimers that were well behavedin solution, whereas 741F8 sFv and sFv' exhibited considerableself-association. The 741F8 sFv binds to the extracellular domain(ECD) of the c-erbB-2 oncogene protein, which is often overexpressedin breast cancer and other adenocarcinomas. The recombinantECD was prepared to facilitate the analysis of 741F8 bindingsite properties; the cloned ECD gene, modified to encode a C-terminalSer-Gly-His6 peptide, was transfected into Chinese hamster ovarycells using a vector that also expressed dihydrofolate reductaseto facilitate methotrexate amplification. Optimized cell linesexpressed ECD-His6 at high levels in a cell bioreactor; afterisolation by immobilized metal affinity chromatography, finalECD yields were as high as 47 mg/l. An animal tumor model complementedphysicochemical studies of 741F8 species and indicated increasedtumor localization of the targeted 741F8 (sFv')2 over othermonovalent 741F8 species. 相似文献
13.
Bell Stephen G.; Harford-Cross Charles F.; Wong Luet-Lok 《Protein engineering, design & selection : PEDS》2001,14(10):797-802
The protein engineering of CYP enzymes for structureactivitystudies and the oxidation of unnatural substrates for biotechnologicalapplications will be greatly facilitated by the availabilityof functional, whole-cell systems for substrate oxidation. Wereport the construction of a tricistronic plasmid that expressesthe CYP101 monooxygenase from Pseudomonas putida, and its physiologicalelectron transfer co-factor proteins putidaredoxin reductaseand putidaredoxin in Escherichia coli, giving a functional invivo catalytic system. Wild-type CYP101 expressed in this systemefficiently transforms camphor to 5-exo-hydroxycamphor withoutfurther oxidation to 5-oxo-camphor until >95% of camphorhas been consumed. CYP101 mutants with increased activity forthe oxidation of diphenylmethane (the Y96FI395G mutant),styrene and ethylbenzene (the Y96FV247L mutant) havebeen engineered. In particular, the Y96FV247L mutantshows coupling efficiency of approximately 60% for styrene andethylbenzene oxidation, with substrate oxidation rates of approximately100/min. Escherichia coli cells transformed with tricistronicplasmids expressing these mutants readily gave 100-mg quantitiesof 4-hydroxydiphenylmethane and 1-phenylethanol in 2472h. This new in vivo system can be used for preparative scalereactions for product characterization, and will greatly facilitatedirected evolution of the CYP101 enzyme for enhanced activityand selectivity of substrate oxidation. 相似文献
14.
为进一步改造细胞色素P450 BM-3酶对吲哚的羟基化能力,以P450 BM-3结构与功能关系的推测为指导,选择突变酶P450 BM-3 (A74G/F87V/L188Q/E435T)为父本,在可能影响P450 BM-3催化吲哚羟基化区域选择性的D168位点进行定点饱和突变,根据全细胞催化产物颜色及组成进行筛选,得到了产物组成、酶动力学性质与父本不同的两个突变酶。突变酶D168W的吲哚羟基化产物中90%是靛玉红,而另一个突变酶D168R的产物中87%是靛蓝,产物组成均不同于亲本。在催化吲哚羟基化时,D168W的kcat与父本相当,但Km却是父本的4.8倍,催化活力只有父本的20%;而D168R的kcat是父本的1.9倍,Km是父本的82%,催化活力比父本提高了1.37倍。结果表明,在E435T突变上叠加D168位氨基酸残基突变对酶的催化性质产生了单一位点突变所不具有的协同效应,对酶催化的区域选择性和催化活力都有显著影响,以致改变了催化产物组成。这种基于知识的半理性定向进化方法由于是在关键位点进行突变,因此突变目的性强、突变效果显著。 相似文献
15.
Wang Xing-guo; Britton K.Linda; Baker Patrick J.; Martin Stephen; Rice David W.; Engel Paul C. 《Protein engineering, design & selection : PEDS》1995,8(2):147-152
Two residues, K89 and S380, thought to interact with the -carboxylgroup of the substrate L-glutamate, have been altered by site-directedmutagenesis of clostridial glutamate dehydrogenase (GDH). Thesingle mutants K89L and S380V and the combined double mutantK89L/S380V were constructed. All three mutants were satisfactorilyoverproduced in soluble form. However, only the K89L mutantwas retained by the dye column normally used in purifying thewild-type enzyme. All three mutant enzymes were purified tohomogeneity and tested for substrate specificity with 24 aminoacids. The single mutant S380V showed no detectable activity.The alternative single mutant K89L showed an activity towardsL-glutamate that was decreased nearly 2000-fold compared withwild-type enzyme, whereas the activities towards the monocarboxylicsubstrates -aminobutyrate and norvaline were increased 2- to3-fold. A similar level of activity was obtained with methionine(0.005 U/mg) and norleucine (0.012 U/mg), neither of which giveany activity with the wild-type enzyme under the same conditions.The double mutant showed decreased activity with all substratescompared with the wild-type GDH. In view of its novel activities,the K89L mutant was investigated in greater detail. A strictlylinear relationship between reaction velocity and substrateconcentration was observed up to 80 mM L-methionine and 200mM L-norleucine, implying very high Km values. Values of kcat/Km,for L-methionine and L-norleucine were 6.7x102 and 0.15s1M1, respectively. Measurements with dithiobisnitrobenzoicacid showed that the mutant enzymes all reacted with a stoichiometryof one -SH group per subunit and all showed protection by coenzyme,indicating essentially unimpaired coenzyme binding. With glutamateor 2-oxoglutarate as substrate the Km values for the vestigialactivity in the mutant enzyme preparations were strikingly closeto the wild-type Km values. Both for wild-type GDH and K89L,L-glutamate gave competitive product inhibition of 2-oxoglutaratereduction but did not inhibit the reduction of 2-oxocaproatecatalysed by K89L enzyme. This suggests that the low levelsof glutamate/2-oxoglutarate activity shown by the mutant enzymeare due to trace contamination. Since stringent precautionswere taken, it appears possible that this reflects the levelof reading error during overexpression of the mutant proteins.CD measurements indicate that the S380V mutant has an alteredconformation, whereas the K89L enzyme gave an identical CD spectrumto that of wild-type GDH; the spectrum of the double mutantwas similar, although somewhat altered in intensity. The resultsconfirm the key role of K89 in dicarboxylate recognition byGDH. 相似文献
16.
Peters I.D.; Hew C.L.; Davies P.L. 《Protein engineering, design & selection : PEDS》1989,3(2):145-151
A semisynthetic winter flounder antifreeze proprotein (proAFP)coding region was constructed and inserted into a lacZ expressionvector. ProAFP was produced from the vector in Escherichia colias a C-terminal fusion to the first 289 amino acids of ß-galactosidase(ß-gal). The proAFP and ß-gal domains ofthe ß-galproAFP fusion protein were separatedby the recognition signal for the blood coagulation protease,factor Xa. Upon induction with isopropylthio-ß-D-galactosidethe fusion protein accumulated to levels of 15% of the totalprotein. The ß-galproAFP fusion protein waspartially purified by differential centrifugation, but requiredsolubilization prior to factor Xa digestion. The solubilizedfusion protein was efficiently and correctly cleaved by factorXa, after which the proAFP was purified by gel permeation. BacterialproAFP was indistinguishable from natural proAFP by the criteriaof antifreeze activity, amino-terminal sequence (15 cycles),reverse-phase HPLC and SDSpolyacrylamide gel electrophoresis.Circular dichroism measurements showed that proAFP is a compositeof random coil and -helical secondary structure, with an -helicalcontent of 44% at 0°C. It seems probable that the C-terminalregion of proAFP, which corresponds to the mature AFP protein,is mainly -helical, and that the N-terminal pro-segment is randomcoiled. 相似文献
17.
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
18.
Nakatani Tomoyuki; Lone Yu-Chun; Yamakawa Junko; Kanaoka Masaharu; Gomi Hideyuki; Wydenes John; Noguchi Hiroshi 《Protein engineering, design & selection : PEDS》1994,7(3):435-443
Mouse monoclonal antihuman IL2 receptor antibody(BB10) inhibits EL2dependent human Tcellproliferation. It has been used in clinical trials in the transplantationfield and promising results are being accumulated. Mouse BB10antibody was humanized by grafting all CDRs and some frameworkamino add residues onto human antibodies, KAS for VH and PAYfor Vx. Nine humanized BBlOs with differently graftedframework residues were constructed and assessed for their biologicalactivities. One of these humanized BB10, M5, showed nearlythe same activity as the mouse BB10. The 49th residueof Vx was demonstrated to play a crucial role in the antigenantibodyinteraction by 3D structure analysis with a computermodeling system. 相似文献
19.
Making tissue-type plasminogen activator more fibrin specific 总被引:2,自引:0,他引:2
Paoni Nicholas F.; Chow Alice M.; Pena Luis C.; Keyt Bruce A.; Zoller Mark J.; Bennett William F. 《Protein engineering, design & selection : PEDS》1993,6(5):529-534
The fibrin specificity of tissue-type plasminogen activatorcan be increased by mutagenesis within at least four sites inthe protease domain. These sites include residue I276, the newN-terminus formed by conversion to a two-chain structure, residueson either side of the active site cleft, KHRR 296299or DDD 364366, a charged surface involved in fibrin interactions,which includes residues H432, R434, D460, R462 and a loop structure,PQANL 466470, near the fibrin-binding patch. Variantswith mutations at any of these sites have low fibrinogen-stimulatedactivity, whereas fibrin-stimulated activity is at least normal.Kinetic analysis reveals that mutations at these positions reducethe kcat in the presence of fibrinogen, but leave the moleculeswith normal kinetic constants in the presence of fibrin. A significantexception is found at positions 296299, where the presenceof fibrin manifests significant increases in both kcat and Km.Combinations of mutations at these sites appear to be additivewith respect to fibrin specificity. 相似文献
20.
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