共查询到20条相似文献,搜索用时 31 毫秒
1.
Smallshaw Joan E.; Georges Fawzy; Lee Jeremy S.; Waygood E.Bruce 《Protein engineering, design & selection : PEDS》1999,12(7):623-630
The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptideheavy chainlinkerlightchain(His)5 tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 1030% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with Kd values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay. 相似文献
2.
Li Jian Yi; Sugimura Keijiro; J.Boado Ruben; Lee Hwa Jeong; Zhang Crystal; Duebel Stefan; M.Pardridge William 《Protein engineering, design & selection : PEDS》1999,12(9):787-796
A single chain Fv antibodystreptavidin fusion proteinwas expressed and purified from bacterial inclusion bodies followingcloning of the genes encoding the variable region of the heavychain and light chain of the murine OX26 monoclonal antibodyto the rat transferrin receptor. The latter undergoes receptormediated transcytosis through the brain capillary endothelialwall in vivo, which makes up the bloodbrain barrier (BBB);therefore, the OX26 monoclonal antibody and its single chainFv analog may act as brain drug delivery vectors in vivo. Attachmentof biotinylated drugs to the antibody vector is facilitatedby production of the streptavidin fusion protein. The bi-functionalityof the OX26 single chain Fv antibodystreptavidin fusionprotein was retained, as the product both bound biotin and therat transferrin receptor in vitro and in vivo, based on pharmacokineticand brain uptake analyses in anesthetized rats. The attachmentof biotinpolyethyleneglycolfluorescein to theOX26 single chain Fv antibodystreptavidin fusion proteinresulted in illumination of isolated rat brain capillaries inconfocal fluorescent microscopy. In conclusion, these studiesdemonstrate that genetically engineered single chain Fv antibodystreptavidinfusion proteins may be used for non-invasive neurotherapeuticdelivery to the brain using endogenous BBB transport systemssuch as the transferrin receptor. 相似文献
3.
Delagrave Simon; Catalan John; Sweet Charles; Drabik Glenn; Henry Andrew; Rees Anthony; Monath Thomas P.; Guirakhoo Farshad 《Protein engineering, design & selection : PEDS》1999,12(4):357-362
HNK20 is a mouse monoclonal IgA that binds to the F glycoproteinof respiratory syncytial virus (RSV) and neutralizes the virus,both in vitro and in vivo. The single-chain antibody fragment(scFv) derived from HNK20 is equally active and has allowedus to assess rapidly the effect of mutations on affinity andantiviral activity. Humanization by variable domain resurfacingrequires that surface residues not normally found in a humanFv be mutated to the expected human amino acid, thereby eliminatingpotentially immunogenic sites. We describe the constructionand characterization of two humanized scFvs, hu7 and hu10, bearing7 and 10 mutations, respectively. Both molecules show unalteredbinding affinities to the RSV antigen (purified F protein) asdetermined by ELISA and surface plasmon resonance measurementsof binding kinetics (Ka 1x109 M1). A competition ELISAusing captured whole virus confirmed that the binding affinitiesof the parental scFv and also of hu7 and hu10 scFvs were identical.However, when compared with the original scFv, hu10 scFv wasshown to have significantly decreased antiviral activity bothin vitro and in a mouse model. Our observations suggest thatbinding of the scFv to the viral antigen is not sufficient forneutralization. We speculate that neutralization may involvethe inhibition or induction of conformational changes in thebound antigen, thereby interfering with the F protein-mediatedfusion of virus and cell membranes in the initial steps of infection. 相似文献
4.
The facile detection and purification of a recombinant proteinwithout detailed knowledge about its individual biochemicalproperties constitutes a problem of general interest in proteinengineering. The use of a novel kind of random peptide libraryfor the stepwise engineering of a C-terminal fusion peptidewhich confers binding activity towards streptavidin is describedin this study. Because of its widespread use as part of a varietyof conjugates and other affinity reagents, streptavidin constitutesthe binding partner of choice both for detection and purificationpurposes. The streptavidin-affinity tag was engineered at theC-terminus of the VH domain as part of the D1.3 Fv fragmentwhich was functionally expressed in Escherichia coli. Irrespectiveof whether it was displayed by the VH or the VL domain, theoptimized version of the affinity peptide termed Strep-tagallowed the detection of the Fv fragment both on Western blotsand in ELISAs by a streptavidinalkaline phosphatase conjugate.In addition, the one-step purification of the intact Fv fragmentcarrying a single Strep-tag at the C-terminus of only one ofits domains was achieved by affinity chromatography with streptavidin-agaroseusing very mild elution conditions. 相似文献
5.
An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli
Takkinen Kristiina; Laukkanen Marja-Leena; Sizmann Dorothea; Alfthan Kaija; Immonen Tiina; Vanne Liisa; Kaartinen Matti; Knowles Jonathan K.C.; Teeri Tuula T. 《Protein engineering, design & selection : PEDS》1991,4(7):837-841
Single-chain antibodies consist of the variable, antigen-bindingdomains of antibodies joined to a continuous polypeptide bygenetically engineered peptide linkers. We have used the flexibleinterdomain linker region of a fungal cellulase to link togetherthe variable domains of an anti-2-phenyloxazolone IgGl and showhere that the resulting single-chain antibody is efficientlysecreted and released to the culture medium of Escherichia coli.The yield of affinity-purified single-chain antibody is 1 -2mg/1 of culture medium and its affinity and stability are comparableto those of the corresponding native IgG. 相似文献
6.
Daugherty Patrick S.; Olsen Mark J.; Iverson Brent L.; Georgiou George 《Protein engineering, design & selection : PEDS》1999,12(7):613-621
Polypeptide library screening technologies are critically dependentupon the characteristics of the expression system employed.A comparative analysis of the lpplac, tet and araBAD promoterswas performed to determine the importance of tight regulationand expression level in library screening applications. Thesurface display of single-chain antibody (scFv) in Escherichiacoli as an LppOmpA' fusion was monitored using a fluorescentlytagged antigen in conjunction with flow cytometry. In contrastto the lpplac promoter, both tet and araBAD promoterscould be tightly repressed. Tight regulation was found to beessential for preventing rapid depletion of library clones expressingfunctional scFv and thus for maintaining the initial librarydiversity. Induction with subsaturating inducer concentrationsyielded mixed populations of uninduced and fully induced cellsfor both the tet and araBAD expression systems. In contrast,homogeneous expression levels were obtained throughout the populationusing saturating inducer concentrations and could be adjustedby varying the induction time and plasmid copy number. Underoptimal induction conditions for the araBAD system, proteinexpression did not compromise either cell viability or librarydiversity. This expression system was used to screen a libraryof random scFv mutants specific for digoxigenin for clones exhibitingimproved hapten dissociation kinetics. Thus, an expression systemhas been developed which allows library diversity to be preservedand is generally applicable to the screening of E.coli surfacedisplayed libraries. 相似文献
7.
Alfthan Kaija; Takkinen Kristiina; Sizmann Dorothea; Soderlund Hans; Teeri Tuula T. 《Protein engineering, design & selection : PEDS》1995,8(7):725-731
Single-chain antibodies were constructed using six differentlinker peptides to join the VH and VL domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)3 linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcoreTM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric. 相似文献
8.
Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein--single-chain antibody fusion protein 总被引:2,自引:0,他引:2
Laukkanen Marja-Leena; Teeri Tuula T.; Keinanen Kari 《Protein engineering, design & selection : PEDS》1993,6(4):449-454
In order to achieve a stable and functional immobilization ofantibodies, we investigated the possibility of adding hydrophobicmembrane anchors to antibody fragments expressed in Escherichiacoli. The DNA sequence encoding the signal peptide and the nineN-terminal amino add residues of the major lipoprotein of E.coliwas fused to the sequence of an anti-2-phenyloxazolone single-chainFv antibody fragment [Takkinen et al. (1991) Protein Engng,4, 837841]. The expression of the fusion construct inE.coli resulted in specific accumulation of an immunoreactive28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment,the fusion protein was cell-associated, labelled by [3H]palmitatewhich is indicative of the presence of N-terminal lipid modification,partitioned into the detergent phase upon Triton X-114 phaseseparation and was localized predominantly in the bacterialouter membrane. The fusion antibody displayed specific 2-phenyloxazolone-bindingactivity in the membranebound form and after solubilizationwith non-ionic detergents. Furthermore, upon removal of detergentthe fusion antibody was incorporated into proteoliposomes whichdisplayed specific hapten-binding activity. Our results showthat antibodies can be converted to membrane-bound proteinswith retention of antigen-binding properties by introductionof lipid anchors during biosynthesis. This approach may proveuseful in the design of immunoliposomes and immunosensors. 相似文献
9.
Saito Akiko; Honda Shinkichi; Nishi Tatsunari; Koike Masamichi; Okazaki Kei; Itoh Seiga; Sato Moriyuki 《Protein engineering, design & selection : PEDS》1989,2(6):481-487
A gene coding for one of the IgG-binding domains of Staphylococcalprotein A, designated domain B, was chemically synthesized.This gene was tandemly repeated to give dimeric and tetramericdomain B genes by the use of two restriction enzymes which gaveblunt ends. The genes were highly expressed in Escherichia colito afford a large amount of dimeric and tetrameric domain Bproteins. The single domain B protein was efficiently producedas a fusion protein with a salmon growth hormone fragment. Thefusion protein was converted to monomeric domain B by cyanogenbromide cleavage. The CD spectra of the monomeric, dimeric andtetrameric domain B proteins were essentially the same as thatof native form protein A, showing that their secondary structureswere very similar. The dimeric and tetrameric domain B proteinsformed precipitates with IgG as protein A. This system permitsthe efficient production of mutated single and multiple IgG-bindingdomains which can be used to study structural changes and proteinAimmunoglobulin interactions. 相似文献
10.
Hinchman Susan K.; Schuster Sheldon M. 《Protein engineering, design & selection : PEDS》1992,5(3):279-283
In order to explore the structure-function relationship of theEscherichia coli asparagine synthetase A it was necessary todevise a system for overexpression of the gene and purificationof the gene product. The E.coli asparagine synthetase A structuralgene was fused to the 3' end of the human carbonic anhydraseII structural gene and overexpressed in E.coli. The gene product,a 66 kDa fusion protein, which exhibited asparagine synthetaseactivity, was purified in a single step by affinity chromatographyand used as the antigen for the production of monoclonal antibodies.The monoclonal antibodies were screened by ELISA. Colonies werechosen which were positive for purified fusion protein and negativefor purified human carbonic anhydrase II. The E.coli asparaginesynthetase A gene was then overexpressed and the gene productwas used without purification for the final screen. The antibodiesselected were used for immunoaffinity chromatography to purifythe recombinant overexpressed E.coli asparagine synthetase A.Thus, a procedure is now available so that asparagine synthetaseA can be purified to homogeneity in a single step. 相似文献
11.
Tripet Brian; Yu Lei; Bautista Daisy L.; Wong Wah Y.; Irvin Randall T.; Hodges Robert S. 《Protein engineering, design & selection : PEDS》1996,9(11):1029-1042
Using the techniques of genetic engineering and the principlesof protein de novo design, we have developed a unique affinitymatrix protein tag system as a rapid, convenient and sensitivemethod to detect, purify and characterize newly expressed recombinantpeptides or proteins from cell extracts. The method utilizestwo de novo-designed linear peptide sequences that can selectivelydimerize to form the stable protein motif, the two-stranded-helical coiled-coil. In this method, a recombinant bacterialexpression vector pRLDE has been engineered so that one of thedimerization strands (E-coil) is expressed as a C-terminal fusiontag on newly expressed peptides or proteins, while the other(K-coil) is either biotin-labeled for detection in a Westernblot-type format or immobilized on an insoluble silica supportfor selective dimerization affinity chromatography. Recombinantlyexpressed peptides from Escherichia coli containing the dimerizationtag have been produced, detected and purified using this method.The recombinant peptides were easily and clearly identifiedusing the biotin-labeled coil, while the single-step affinitypurification results indicated the purity of the affinity purifiedexpressed peptides to be >95%, as assessed by reversed-phasechromatography. The stability of the dimerization domain alsoallows for the purified peptide to be left attached to the matrix,thus creating a new peptide-bound column that can be used tostudy peptideprotein or peptideligand interactions.Therefore this system offers a new alternative to existing peptideor protein fusion tags and demonstrates the utility of a denovo-designed system. 相似文献
12.
Field Helen; Yarranton G.T.; Rees A.R. 《Protein engineering, design & selection : PEDS》1990,3(7):641-647
The expression of immunoglobulin heavy and light chain variableregions in the cytoplasm of Escherichia coli and formation ofa functional heterodimer has been demonstrated. Variable domainsequences were taken from the heavy and light chain cDNAs ofthe monoclonal antibody Gloop 2 and engineered for expressionin a dual origin expression vector. The engineered genes vhg2and vlg2 were separately subcloned into the vector, creatingtwo expression plasmids. Expression of the heavy and light chainvariable region genes (encoding 116 and 109 amino adds respectively)was investigated in eight E.coli strains; the polypeptides wererapidly degraded in a host strain optimized for expression andin E.coli strains deficient in the major protease La (lon-).Accumulation was permitted in severely protease-deficient E.colihaving a defective heat-shock response. A lon- mutation in thisgenetic background permitted even higher accumulation. Expressionlevels were 7 and 1% of total bacterial protein for light andheavy chain variable regions respectively. Expression of theheavy chain variable region gene was increased by includinga longer ShineDalgarno sequence. Similar constructionsin the light chain vector had no effect on expression levels.The insoluble variable region polypeptides were reconstitutedinto a heterodimer possessing the full antigen binding characteristicsof both the parent monoclonal antibody and its Fab fragment. 相似文献
13.
Green fluorescent antibodies: novel in vitro tools 总被引:2,自引:0,他引:2
Casey Joanne L.; Coley Andrew M.; Tilley Leann M.; Foley Michael 《Protein engineering, design & selection : PEDS》2000,13(6):445-452
We produced a fluorescent antibody as a single recombinant proteinin Escherichia coli by fusing a red-shifted mutant of greenfluorescent protein (EGFP) to a single-chain antibody variablefragment (scFv) specific for hepatitis B surface antigen (HepBsAg).GFP is a cytoplasmic protein and it was not previously knownwhether it would fold correctly to form a fluorescent proteinin the periplasmic space of E.coli. In this study we showedthat EGFP alone or fused to the N'- and C'-termini of the scFvresulted in fusion proteins that were in fact highly fluorescentin the periplasmic space of E.coli cells. Further characterizationrevealed that the periplasmic N'-terminal EGFPscFv fusionwas the most stable form which retained the fluorescent propertiesof EGFP and the antigen binding properties of the native scFv;thus representing a fully functional chimeric molecule. We alsodemonstrated the utility of EGFPscFv in immunofluorescencestudies. The results showed positive staining of COS-7 cellstransfected with HepBsAg, with comparable sensitivity to a monoclonalantibody or the scFv alone, probed with conventional fluorescein-labelledsecond antibodies. In this study, we developed a simple techniqueto produce fluorescent antibodies which can potentially be appliedto any scFv. We demonstrated the utility of an EGFPscFvfusion protein for immunofluorescence studies, but there aremany biological systems to which this technology may be applied. 相似文献
14.
Directing phage selections towards specific epitopes 总被引:1,自引:0,他引:1
Parsons Helen L.; Earnshaw John C.; Wilton Jane; Johnson Kevin S.; Schueler Paula A.; Mahoney Walt; McCafferty John 《Protein engineering, design & selection : PEDS》1996,9(11):1043-1049
It is possible to direct selections from antibody repertoiresdisplayed on filamentous phage towards unique epitopes on proteinantigens by competing with related molecules. A phage displayrepertoire of human single chain Fvs (scFvs) was panned threetimes against foetal haemoglobin (HbF). The selection was dominatedby one clone with a Kd of 10 nM but yielded at least 17 others,all of which bound HbF but crossreacted with adult haemoglobin(HbA). To direct selection towards HbF-specific epitopes, therepertoire was preincubated with HbA in solution before eachpanning. Crossreactive scFvs can form complexes with the solubleHbA and thereby be prevented from binding the immobilized HbF.Four clones with preferential binding to HbF emerged under theseconditions. One of these (Hb-1), with a Kd of 6 µM, hadexquisite specificity for HbF and could distinguish cells expressingHbF from those expressing HbA by immunocytochemistry and flowcytometry. This antibody has an affinity that is 600-fold lowerthan the dominant crossreactive clone, and so only emerged underconditions of competitive deselection. Thus, competitivedeselection is a viable means for directing selections towardsuseful epitopes. It permits a more effective searchof phage display repertoires and allows the emergence of loweraffinity clones with useful specificities. These clones maybe useful in themselves or may serve as leads for in vitro affinitymaturation. 相似文献
15.
The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolatedehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolatesynthetase was engineered to contain a prokaryotic ribosomebinding site and was expressed under the bacteriophage T7 RNApolymerase promoter in Escherichia coli. Site-directed mutagenesiswas used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase(D/C) domain as amino acid residues 1301, and the synthetase(Syn) domain as residues 304935. Both domains formedactive enzymes thereby demonstrating their ability to fold independently.The full-length enzyme, D/C and Syn domains were expressed atlevels 4-, 55- and 3-fold higher than the specific activitiesfound in liver. Additional mutagenesis and independent expressionof domains further defined the interdomain region to includeamino acids 292310. The D/C domain was purified to homogeneityby a single affinity chromatographic step, and the full-lengthprotein in a twostep procedure. The kinetic properties of theD/C domain appear unaltered from those of the trifunctionalenzyme. 相似文献
16.
Jennings P.A.; Bills M.M.; Irving D.O.; Mattick J.S. 《Protein engineering, design & selection : PEDS》1989,2(5):365-369
The pattern of sequence variation between Bacteroides nodosusfimbrial subuits of different serotypes suggests a degree offlexibility, which might be exploited for protein engineeringapproaches for the expression of other peptides. We have testedthis using the well-characterized peptide epitope from VP1 offoot-and-mouth disease virus (FMDV), residues 144159:LRGDLQVLAQKVARTL (strain 01-BFS). Using bacterial codon usage,several oligonucleotides were designed for the substitutionof this sequence internally at hypervariable regions of thefimbrial subunit (aligned for maximum homology), and for itsaddition at the carboxy-terminus with a diglycine spacer asa flexible hinge. Following site-directed mutagenesis in phageM13, the modified genes were placed under PL promoter controland placed in a broad host range vector. Analysis of the variantproteins expressed in E.coli showed that these substitutionsaffected, to varying extents, recognition by both anti-fimbrialand anti-FMDV antibodies, indicating that hypervariable region2 is a major antigenic determinant of the fimbrial subunit andthat local stereochemical effects can influence antibody bindingto the FMDV peptide antigenic determinant. In Pseudomonas aeruginosa,viable transformants could only be obtained with the mutantgene encoding the carboxy-terminal graft. These cells providedfimbrial preparations comprised of the modified subunit. Thisthen constitutes the prototype for the development of a generalexpression system for the production of vaccine epitopes andother bioactive peptides. Furthermore, there is considerablescope for further modification of the system, for example byengineering specific chemical or protease cleavage sites forrelease of the grafted peptide. Alternatively, the fimbriaethemselves may serve as a useful supramoleuclar carrier or adjuvantfor immune provocation. 相似文献
17.
Guillemette J.Guy; Matsushima-Hibiya Yuko; Atkinson Tom; Smith Michael 《Protein engineering, design & selection : PEDS》1991,4(5):585-592
A gene for expression of horse heart myoglobin in Escherichiacoli has been constructed in one step from long synthetic oligonucleotides.The synthetic gene contains an efficient translation initiationsignal and used codons that are commonly found in E.coli. Uniquerestriction sites are placed throughout the gene. It has beeninserted in a phagemid vector and is expressed from the lacpromoter in E.coli at high efficiency, the soluble heme proteinrepresenting 10% of soluble protein. Two versions of horse heartmyoglobin were produced with aspartic acid or asparagine atresidue 122. Comparison of chromatographic mobilities of thesetwo proteins with authentic horse heart myoglobin identifiedaspartic acid as the correct residue 122. The availability ofthis gene, which is designed to facilitate oligonucleotide mutagenesisor cassette mutagenesis, will allow systematic structurefunctionanalysis of horse heart myoglobin. 相似文献
18.
Directed evolution for the development of conformation-specific affinity reagents using yeast display 总被引:1,自引:0,他引:1
Weaver-Feldhaus JM Miller KD Feldhaus MJ Siegel RW 《Protein engineering, design & selection : PEDS》2005,18(11):527-536
Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca(2+) binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca(2+)-bound (Ca(2+)-CaM) or Ca(2+)-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (K(d) = 0.8 nM) and specificity (>1000-fold) for Ca(2+)-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (K(d) = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform. 相似文献
19.
Schwede Torsten F.; Badeker Mathias; Langer Martin; Retey Janos; Schulz Georg E. 《Protein engineering, design & selection : PEDS》1999,12(2):151-153
Histidase (histidine ammonia-lyase, EC 4.3.1.3) from Pseudomonasputida was expressed in Escherichia coli and purified. In theabsence of thiols the tetrameric enzyme gave rise to undefinedaggregates and suitable crystals could not be obtained. Thesolvent accessibility along the chain was predicted from theamino acid sequence. Among the seven cysteines, only one waslabeled as `solvent-exposed'. The exchange of this cysteineto alanine abolished all undefined aggregations and yieldedreadily crystals diffracting to 1.8 Å resolution. 相似文献
20.
Dekeyzer N.; Engelborghs Y.; Volckaert G. 《Protein engineering, design & selection : PEDS》1994,7(1):125-130
A gene coding for the Nereis sarcoplasmic calcium-binding protein(NSCP) was synthesized and expressed in Escherichia coli. Thesequence of the gene was derived from the protein sequence byreverse translation. It possesses a number of unique, regularlyspaced, restriction endonuclease cleavage sites to facilitatefuture site-directed mutagenesis. For the cloning strategy thegene sequence was divided into four parts. Three parts werecloned by ligation of hybridized oligomers and one part by inversePCR. The protein was expressed as a fusion protein with thebacterial chloramphenicol acetyltransferase (CAT), which couldbe easily purified by affinity chromatography. At the junctionof the CAT and NSCP moieties a recognition site for the proteolyticenzyme factor Xa was built in. However, the distance betweenthe moieties appeared to be crucial to warrant cleavage. A kineticanalysis showed that NSCP prepared from the sandworm and theone expressed by E.coli behaved in the same way. This systemprovides a basis for site-specific mutagenesis studies, in orderto elucidate the molecular mechanism of cation binding and concomitantconformational changes 相似文献