共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
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
3.
Cols Neus; Romero-Isart Nuria; Bofill Roger; Capdevila Merce; Gonzalez-Duarte Pilar; Gonzalez-Duarte Roser; Atrian Silvia 《Protein engineering, design & selection : PEDS》1999,12(3):265-269
The ß domain of mouse metallothionein 1 (ßMT) wassynthesized in Escherichia coli cells grown in the presenceof copper or cadmium. Homogenous preparations of CußMTand CdßMT were used to characterize the correspondingin vivo-conformed metal-clusters, and to compare them with thespecies obtained in vitro by metal replacement to a canonicalZn3ßMT structure. The copper-containing ßMTclusters formed inside the cells were very stable. In contrast,the nascent ß peptide, although it showed cadmium bindingability, produced a highly unstable species, whose stoichiometrydepended upon culture conditions. The absence of ßMT proteinin E.coli protease-proficient hosts grown in cadmium-supplementedmedium pointed to drastic proteolysis of a poorly folded ßpeptide, somehow enhanced by the presence of cadmium. Possiblefunctional and evolutionary implications of the bioactivityof mammalian ßMT in the presence of monovalent and divalentmetal ions are discussed. 相似文献
4.
Engels J.W.; Glauder J.; M?llner H.; Tripier D.; Uhlmann E.; Wetekam W. 《Protein engineering, design & selection : PEDS》1987,1(3):195-199
By chemoenzymatic synthesis the gene for a (Leu27) analogueof human growth hormone releasing hormone-Gly45 [(Leu27GHRH-Gly45]was constructed, cloned and expressed in Escherichia coli asa fusion protein with ß-galactosidase under the controlof the lac promoter and operator. Upon induction with isopropyl-D-thio-ß-galactopyranosidethe fusion protein accumulated to a yield of 1520% ofthe total cellular protein. After cyanogen bromide deavage ofthe fusion protein the precursor peptide (Leu27)hGHRH-Gly45was separated by extraction and purified by ion exchange andh.p.l.c.-RP18 chromatography. The purified peptide was analysedby sequencing, isolectric focusing, amino acid analysis andamino acid analysis after V8 protease digestion. The carboxy-terminalglydne was subsequently amidated by PAM (peptidylglycine--amidating-monooxygenase),an enzyme which was isolated and characterized from fresh bovinepituitaries. Correct amidatlon of the penultimate amino acid,leucine, was verified by peptide sequencing with an authenticleucine amide reference. 相似文献
5.
Kankare Jussi; Neal Genevieve S; Salminenlr Tiina; Glumhoff Tuomo; Cooperman Barry S; Lahti Reijo; Goldman Adrian 《Protein engineering, design & selection : PEDS》1994,7(7):823-830
Hie structure of E.coli soluble inorganic pyrophosphatase hasbeen refined at 2.7 resolution to an R-factor of 20.9. Theoverall fold of the molecule is essentially the same as yeastpyrophosphatase, except that yeast pyrophosphatase is longerat both the N- and C-termini. Escherichia coli pyrophosphataseis a mixed +ß protein with a complicated topology.The active site cavity, which is also very similar to the yeastenzyme, is formed by seven ß-strands and an -helixand has a rather asymmetric distribution of charged residues.Our structure-based alignment extends and improves upon earliersequence alignment studies; it shows that probably no more than14, not 1517 charged and polar residues are part of theconserved enzyme mechanism of pyrophosphatases. Six of theseconserved residues, at the bottom of the active site cavity,form a tight group centred on Asp70 and probably bind the twoessential Mg+ ions. The others, more spreadout and more positivelycharged, presumably bind substrate. Escherichia coli pyrophosphatasehas an extra aspartate residue in the active site cavity, whichmay explain why the two enzymes bind divalent cation differently.Based on the structure, we have identified a sequence motifthat seems to occur only in soluble inorganic pyrophosphatases. 相似文献
6.
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
7.
Finkelstein Alexei V.; Nakamura Haruki 《Protein engineering, design & selection : PEDS》1993,6(4):367-372
A lack of knowledge about the construction of tight packingis now the main obstacle for a successful design of artificialproteins. In this paper we examine a way of close packing antiparallelß-sandwihes. We show that there are some weakpoints at the surfaces of ß-sheets, which cannotbe filled by the surrounding aliphatic side chains that arethe most abundant. Theoretically, these weak pointscan be filled either by aromatic side chains of the same sheetor by the residues of the other parts of the protein molecule.The analysis of protein structures shows that both possibilitiesare used by nature and that there are many cases when theseweak points are not filled by any atom. They remainfree and form a majority of the defects of close packing inprotein globules. 相似文献
8.
Boeggeman Elizabeth E; Balaji Petety V; Sethi Navin; Masibay Arni S; Qasba Pradman K 《Protein engineering, design & selection : PEDS》1993,6(7):779-785
Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90
[EC]
) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an oxido-shufflingreagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 3085%of the sp. act. of bovine milk ß-1, 4-GT with apparentKms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity. 相似文献
9.
By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 812 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg2+. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 2731 of wild-type)resulted in enzymes with greater dependence on Mg2+ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence. 相似文献
10.
Christmann Andreas; Walter Kerstin; Wentzel Alexander; Kratzner Ralph; Kolmar Harald 《Protein engineering, design & selection : PEDS》1999,12(9):797-806
The Ecballium elaterium trypsin inhibitor II (EETI-II), a memberof the squash family of protease inhibitors, is composed of28 amino acid residues and is a potent inhibitor of trypsin.Its compact structure is defined by a triple-stranded antiparallelß-sheet, which is held together by three intramoleculardisulfide bonds forming a cystine knot. In order to explorethe potential of the EETI-II peptide to serve as a structuralscaffold for the presentation of randomized oligopeptides, weconstructed two EETI-II derivatives, where the six-residue inhibitorloop was replaced by a 13-residue epitope of Sendai virus L-proteinand by a 17-residue epitope from human bone Gla-protein. EETI-IIand derived variants were produced via fusion to maltose bindingprotein MalE. By secretion of the fusion into the periplasmicspace, fully oxidized and correctly folded EETI-II was obtainedin high yield. EETI-II and derived variants could be presentedon the Escherichia coli outer membrane by fusion to truncatedLpp'OmpA', which comprises the first nine residues ofmature lipoprotein plus the membrane spanning ß-strandfrom residues 4666 of OmpA protein. Gene expression wasunder control of the strong and tightly regulated tetA promoter/operator.Cell viability was found to be drastically reduced by high levelexpression of Lpp'OmpA'EETI-II fusion protein.To restore cell viability, net accumulation of fusion proteinin the outer membrane was reduced to a tolerable level by introductionof an amber codon at position 9 of the lpp' sequence and utilizingan amber suppressor strain as expression host. Cells expressingEETI-II variants containing an epitope were shown to be surfacelabeled with the respective monoclonal antibody by indirectimmunofluorescence corroborating the cell surface exposure ofthe epitope sequences embedded in the EETI-II cystine knot scaffold.Cells displaying a particular epitope sequence could be enriched107-fold by combining magnetic cell sorting with fluorescence-activatedcell sorting. These results demonstrate that E.coli cell surfacedisplay of conformationally constrained peptides tethered tothe EETI-II cystine knot scaffold has the potential to becomean effective technique for the rapid isolation of small peptidemolecules from combinatorial libraries that bind with high affinityto acceptor molecules. 相似文献
11.
Witt Ellen; Stewart George C.; Hengstenberg Wolfgang 《Protein engineering, design & selection : PEDS》1992,5(3):267-271
The lacG gene encoding the 6-phospho-ß-galactosidase(E.C.3.2.1.85) of Staphylococcus aureus was fused to the proteinA gene in the plasmid pRIT2T. Escherichia coli cells containingthis plasmid produce a fusion protein with both IgG bindingand 6-phospho-ß-galactosidase activities after heatinduction. The recombinant gene was overexpressed and the hybridprotein was purified to homogeneity in high yield. The chimericprotein was shown to have almost identical enzymatic characteristicsto pure 6-phospho-ß-galactosidase. This result leadsto the conclusion that a free N-terminus of the 6-phospho-ß-galactosidaseis not required for biological activity. The hybrid proteinof protein A and 6-phospho-ß-galactosidase was usedas an enzyme conjugate in enzyme-linked immunosorbent assays(ELISA). The experiments presented demonstrate that the 6-phospho-ß-galactosidaseis a suitable fusion partner in various diagnostic applicationswhere an unique biological activity is required. 相似文献
12.
Witt Ellen; Frank Rainer; Hengstenberg Wolfgang 《Protein engineering, design & selection : PEDS》1993,6(8):913-920
The 6-phospho-ß-galactosidase of Staphylococcus aureus,Lactococcus lactis and Lactobacillus casei and 6-phospho-ßglucosidaseB of Escherichia coli build a subfamily inside a greater enzymefamily, named the glycosal hydrolase family 1, which, hi addition,contains nine ß-glycosidases of different origins.Kinetic and immunological evidence is provided in this reportwhich strengthens the relationship of the four 6-phospho-ß-glycosidases.It is shown that the 6-phospho-ß-galactosidases and6-phospho-ß-glucosidase B are able to split aromaticß-galactoside phosphates and ß-glucosidephosphates. The turnover numbers of hydrolysis of substrateswith different epimerization at C-4 of the glycon vary up to15-fold only. Two polydonal antisera, one derived against thenative 6-phospho-ß-galactosidase from S.aureus andthe other derived against the 6-phospho-ß-glucosidaseB, cross-reacted with both enzymes. Peptides of the proteinswere separated by reverse phase HPLC. The cross-reacting peptideswere sequenced and shown to be localized at almost the sameposition in the aligned primary structures of both enzymes.An insertion of nine amino adds near these antigenic domainsis unique for the 6-phospho-ß-glycosidases and missingwithin the sequences of the ß-glycoside-specific membersof the family. The lacG gene of a 6-phospho-ß-galactosidasenegative S.aureus mutant was doned into E.coli and sequenced.In the totally inactive mutant protein only the glycine at position332 was changed to an arginine. This amino acid is part of thesequence insertion near the antigenic domain reacting with bothantisera. These data support the assumption that the regionis of great importance for the function of the enzymes and thatit is possible it determines the specificity of the phosphorylatedform of the substrates. In addition, the 6-phospho-ß-galactosidaseof S.aureus was modified by sitedirected mutagenesis of thecorresponding lacG gene hi order to replace residues Glul60and Glu375, which were suspected of being involved hi the generalacid catalysis of substrate hydrolysis, with glutamine residues.The mutant protein 160EQ retained some catalytic activity whilethe protein 375EQ was totally inactive. Glu375 is the activesite nudeophile of the 6-phospho-ß-galactosidase ofS.aureus. It is located in the sequence motif ENG where Glu358was identified as the catalytkally active nudeophile hi theß-glucosidase of Agrobacterium. 相似文献
13.
In an attempt to introduce a large peptide that is not normallytranslocated across membranes into the cytosol of eukaryoticcells, we created a new chimeric protein termed CEDH betweenPseudomonas aeruginosa exotoxin A (ETA) and a variant enzymeof Mus musculus dihydrofolate reductase (DHFR) with reducedaffinity for antifolates, ETA1413.DHFR1187.ETA609613.We have defined, genetically constructed and expressed the chimericprotein in Escherichia coli. We showed that the CEDH chimericprotein, purified to homogeneity on an immunoaffinity resin,confers a methotrexate-resistant phenotype to Chinese hamsterovary cells. Furthermore, the chimeric protein allowed the growthof dihydrofolate reductase-deficient Chinese hamster ovary cellsin the absence of hypoxanthine and thymidine. These resultsdemonstrated that the chimeric protein exhibited enzyme activityand possessed the tightly folded native structure, and thatthe DHFR protein can be selectively internalized and translocatedvia domains of exotoxin A. These data show that the ETA systemis an efficient system for the delivery of a variety of largepolypeptides into the cytosol without stress to the target cells,and extends the use of this delivery system to proteins thatare not normally translocated across membranes. 相似文献
14.
Replacing the ({beta}{alpha})-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme 总被引:2,自引:0,他引:2
Mainfroid Veronique; Goraj Karine; Rentier-Delrue Francoise; Houbrechts Annick; Noble Martin E.M.; Borchert Torben V.; Wierenga Rik K.; Martial Joseph A. 《Protein engineering, design & selection : PEDS》1993,6(8):893-900
In order to investigate how structural modifications interferewith protein stability, we modified a (ß)-unit inE.coli triosephosphate isomerase (TIM), a typical (ß)-barrelprotein, assuming that the pseudosymmetrical ß-barrelcan be divided into eight successive loop/ß-strand/loop/-helixmotifs. We replaced the eighth (ß)-unit of E.coliTIM with the corresponding chicken (ß)-unit. The substitution,involving the replacement of 10 of the 23 residues of this (ß)-unit, was evaluated first by modelling, then experimentally.Modelling by bomology suggests how the amino add replacementsmight be accommodated in the hybrid E.coli/chicken TIM (ETCM8CHI).Both natural and hybrid recombinant TIMs, overproduced in E.coli,were purified to homogeneity and characterized as to their stabilityand kinetics. Our kinetic studies show that the modificationperformed here leads to an active enzyme. The stability studiesindicate that the stability of ETIM8CHI is comparable to thatof the wild type TIM. 相似文献
15.
Becerra M.; Prado S.Diaz; Siso M.I.Gonzalez; Cerdan M.E. 《Protein engineering, design & selection : PEDS》2001,14(5):379-386
We examined several strategies for the secretion of Kluyveromyceslactis ß-galactosidase into the culture medium, inorder to facilitate the downstream processing and purificationof this intracellular enzyme of great industrial interest. Weconstructed plasmids by fusing the LAC4 gene or engineered variantsto the secretion signal of the K.lactis killer toxin or to thesecretion signal of the Saccharomyces cerevisiae -factor. Withthese plasmids we transformed strains of the yeasts K.lactisand S.cerevisiae, respectively and tested ß-galactosidaseextracellular activity in different culture media. We achievedpartial secretion of ß-galactosidase in the culturemedium since the high molecular weight and oligomeric natureof the enzyme, among other factors, preclude full secretion.The percentage of secretion was improved by directed mutagenesisof the N-terminus of the protein. We developed several deletionmutants which helped us to propose structurefunctionrelationships by comparison with the available data on the homologousEscherichia coli ß-galactosidase. The influence ofthe culture conditions on heterologous ß-galactosidasesecretion was also studied. 相似文献
16.
Ikeda Yutaka; Kawahara Shun-ichi; Taki Masumi; Kuno Atsushi; Hasegawa Tsunemi; Taira Kazunari 《Protein engineering, design & selection : PEDS》2003,16(9):699-706
Proteins containing unnatural amino acids have immense potentialin biotechnology and medicine. We prepared several histidineanalogues including a novel histidine analogue, ß-(1,2,3-triazol-4-yl)-DL-alanine.These histidine analogues were assayed for translational activityin histidine-auxotrophic Escherichia coli strain UTH780. Weobserved that several histidine analogues, including our novelhistidine analogue, were efficiently incorporated into the proteinin vivo; however, other analogues were rejected. These resultssuggest that the hydrogen atom at a specific position seriouslyaffects incorporation. Received April 10, 2003; revised June 20, 2003; accepted July 22, 2003. 相似文献
17.
Zawadzke Laura E.; Smith Tom J.; Herzberg Osnat 《Protein engineering, design & selection : PEDS》1995,8(12):1275-1285
The ß-lactamase from Staphylococcus aureus PCI hasbeen cloned into an Escherichia coli vector for site-directedmutagenesis and high-level protein expression. A mutant enzymehas been produced in which Ala238 is replaced by a serine, andIle239 is deleted (A238S:I239del). The engineered enzyme hydrolysesthird-generation cephalosporins substantially more rapidly thanthe parental enzyme does, while hydrolysis of benzylpenicillinis slower with the mutant than with the wild-type and nativeenzymes. The mutant P-lactamase has been crystallized and thestructure determined and refined at 2.8 A resolution. The dispositionof the ß-strand which forms the side of the activesite is altered in comparison with the native S.aureus ß-lactamasestructure, widening the active site cleft and providing spaceto accommodate the bulky side-chains of the third-generationcephalosporins. 相似文献
18.
Tzakos Andreas G.; Galanis Athanassios S.; Spyroulias Georgios A.; Cordopatis Paul; Manessi-Zoupa Evy; Gerothanassis Ioannis P. 《Protein engineering, design & selection : PEDS》2003,16(12):993-1003
Human somatic angiotensin I-converting enzyme (sACE) has twoactive sites present in two sequence homologous protein domains(ACE_N and ACE_C) possessing several biochemical features thatdifferentiate the two active sites (i.e. chloride ion activation).Based on the recently solved X-ray structure of testis angiotensin-convertingenzyme (tACE), the 3D structure of ACE_N was modeled. Electrostaticpotential calculations reveal that the ACE_N binding grooveis significantly more positively charged than the ACE_C, whichprovides a first rationalization for their functional discrimination.The chloride ion pore for Cl2 (one of the two chloride ionsrevealed in the X-ray structure of tACE) that connects the externalsolution with the inner part of the protein was identified onthe basis of an extended network of water molecules. Comparisonof ACE_C with the X-ray structure of the prokaryotic ClC Clchannel from Salmonella enterica serovar typhimurium demonstratesa common molecular basis of anion selectivity. The criticalrole for Cl2 as an ionic switch is emphasized. Sequence andstructural comparison between ACE_N and ACE_C and of other proteinsof the gluzincin family highlights key residues that could beresponsible for the peptide hydrolysis mechanism. Currentlyavailable mutational and substrate hydrolysis data for bothdomains are evaluated and are consistent with the predictedmodel. Received May 23, 2003; revised October 16, 2003; accepted October 21, 2003 相似文献
19.
Nishiyama Makoto; Suzuki Junko; Ohnuki Tatsuya; Chang Hae Choon; Horinouchi Sueharu; Turley Stewart; Adman Elinor T.; Beppu Teruhiko 《Protein engineering, design & selection : PEDS》1992,5(2):177-184
Pseudoazurin (a blue copper protein or cupredoxin) of a denitrifyingbacterium Alcaligenes faecalis S-6 is a direct electron carrierfor a Cu-containing nitrite reductase (NIR) of the same organism.Site-directed mutagenesis of the pseudoazurin was carried outusing an Escherichia coli expression system. Replacement ofTyr74 by Phe to remove an internal hydrogen bond in the ß-barrelcaused a slight decrease in heat stability as well as a requirementfor a higher concentration of Cu2+ for production in the E.colihost. Exchange of Ala for Pro80 adjacent to His81, one of thefour ligands binding a type I Cu atom, caused a marked increasein reduction potential by 139 mV without change in the opticalabsorption spectrum. The ability of the pseudoazurin to transferelectrons to NIR was markedly diminished but the apparent Kmof NIR for pseudoazurin was not affected by the mutation. X-raydiffraction data collected on the oxidized and reduced formsof the Pro80Ala mutant show that a water molecule occupies thepocket created by the absent side chain. This observation suggeststhat the increase in reduction potential may be caused due tothe increased solvent accessibility to the Cu atom. The electrondensity difference maps on these structures (at 2.0 Å)show that this water moves during the change in oxidation state,and that there are small, but localized, conformational changes>6.5 Å from the copper site, as well as movement ofboth the Cu2+ and the cysteinate sulfur. 相似文献
20.
Heaphy Shaun; Singh Mohinder; Gait Michael J. 《Protein engineering, design & selection : PEDS》1987,1(5):425-431
A synthetic gene coding for the bacteriocidal protein caltrin/seminalplasminwas constructed and expressed in Escherichia coli as a fusionwith ß-galactosidase. The gene was designed with arecognition site for the plasma protease, Factor Xa, coded forimmediately prior to the N-terminus of caltrin. The ß-galactosidase-caltrinfusion protein was cleaved with Factor Xa to give caltrin, whichwas identified by its size on SDS-PAGE, its ability to reactwith an antiserum raised to the N-terminal nonapeptide of caltrinand its N-terminal amino acid sequence. After partial purification,synthetic caltrin was found to be active in an assay involvinginhibition of growth of E.coli. 相似文献