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1.
Belin P Dassa J Drevet P Lajeunesse E Savatier A Boulain JC Ménez A 《Protein engineering, design & selection : PEDS》2004,17(5):491-500
We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding. For an efficient selection, we found that time of toxicity induction must be precisely determined and recombinant protein must be expressed as a fusion with a protein whose activity is easily detectable on plates, thus allowing elimination of non-productive mutants. Choosing the expression to the periplasmic space of an scFv fragment fused to the N-terminus of alkaline phosphatase as a model, we selected chromosomal mutations that reduce aggregation-induced toxicity and showed that they concomitantly improve production of a functional recombinant hybrid. The effects of the mutations isolated could then be cumulated with those of other strategies used for recombinant scFv production. Thus, we could ensure a 6- to 16-fold increase in production of a functional scFv-PhoA hybrid. This is the first report demonstrating the possibility of directly selecting on agar plates E.coli strains improved for functional recombinant protein production from a large bacterial mutant library. 相似文献
2.
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
3.
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
4.
A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli 总被引:1,自引:0,他引:1
Rabhi-Essafi I Sadok A Khalaf N Fathallah DM 《Protein engineering, design & selection : PEDS》2007,20(5):201-209
Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFNalpha2b as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli. 相似文献
5.
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
6.
Williams Neal K.; Peide Yin; Seymour Kristen K.; Ralston Gregory B.; Christopherson Richard I. 《Protein engineering, design & selection : PEDS》1993,6(3):333-340
Dihydroorotase is the central domain of trifunctional L-dihydroorotatesynthetase which also contains carbamyl phosphate synthetaseat the N-terminus and aspartate transcarbamylase at the C-terminus.The cDNA, corresponding to the active dihydroorotase domainas isolated after digestion of dihydroorotate synthetase withelastase, has been sub-cloned into the expression vector pCW12which was then used to transform Escherichia coli SØ1263pyrC lacking dihydroorotase activity. However, inductionof this recomhinant strain with IPTG produced large amountsof the dihydroorotase domain which were completely inactive.A number of cDNAs were expressed which were longer on the C-terminalside; all cDNAs expressed active dihydroorotase domain downto a minimal extension of 12 ammo adds (-Val- Pro-Pro-Gly-Tyr-GIy-Gm-Asp-Val-Arg-Lys-Trp)into the bridge region between the dihydroorotase and aspartatetranscarbamylase domains. Part of this dodecapeptide may forman amphipathk helix which in some way constrains the isolated,recombinant dihydroorotase domain to an active conformation.The recombinant hamster dihydroorotase purified from a cell-freeextract of E.coli in four steps has a turnover number of 297mol/min/(mol domain) for the conversion of L-dihydroorotateback to N-carbamyl-Laspartate with K8 = 8.7 ± 1.5 µMfor L-dihydroorotate, a subunit molecular weight of 39 008 determinedfrom the sequence and 37 900 ± 400 when subjected toSDSPAGE, and an isoelectric point of 5.7. Ultracentrifugalanalysis of the recombinant domain showed a single species ofs20,w = 4.1 S and a single molecular species of Mr = 76 000corresponding to a dimer. 相似文献
7.
8.
Wang Chang-Lin; Huang May; Wesson Carla A.R.; Birdsell Dale C.; Trumble William R. 《Protein engineering, design & selection : PEDS》1994,7(5):715-722
A recombinant gene fusion was created and cloned using a previouslyconstructed gene encoding a monodomain IgG Fc binding proteinand the gene coding for bacterial alkaline phosphatase. Theconstruct was able to express and secrete a fusion protein thatexhibited both IgG binding and alkaline phosphatase enzymaticactivities. Greater than 60% of the protein demonstrating bothbiological activities was detected from periplasmic space preparations.Nanogram concentrations of the Fc binding-alkaline phosphatasefusion protein allowed primary IgG antibody detection withoutthe use of conjugated secondary antibodies. Removal of the domaincoding for alkaline phosphatase resulted in decreased resistanceof the protein to proteolytic degradation and the loss of IgGFc binding ability. Using affinity-purified fusion protein,the specificity of binding to IgG, IgM and IgA was examined;binding was strong to IgG and barely detectable against IgMor IgA. Affinity for binding of the fusion protein to IgG (kd= 6.7 x10-8 M) was determined to be equal to or greater thanpreviously reported for protein A. 相似文献
9.
Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11 总被引:7,自引:0,他引:7
delCardayre Stephen B.; Ribo Marc; Yokel Erich M.; Quirk David J.; Rutter William J.; Raines Ronald T. 《Protein engineering, design & selection : PEDS》1995,8(3):261-273
Bovine pancreatic ribonuclease A (RNase A) has been the objectof much landmark work in biological chemistry. Yet the applicationof the techniques of protein engineering to RNase A has beenlimited by problems inherent in the isolation and heterologousexpression of its gene. A cDNA library was prepared from cowpancreas, and from this library the cDNA that codes for RNaseA was isolated. This cDNA was inserted into expression plasmidsthat then directed the production of RNase A in Saccharomycescerevisiae (fused to a modified -factor leader sequence) orEscherichia coli (fused to the pelB signal sequence). RNaseA secreted into the medium by S.cerevisiae was an active buthighly glycosylated enzyme that was recoverable at 1 mg/l ofculture. RNase A produced by E.coli was in an insoluble fractionof the cell lysate. Oxidation of the reduced and denatured proteinproduced active enzyme which was isolated at 50 mg/l of culture.The bacterial expression system is ideal for the large-scaleproduction of mutants of RNase A. This system was used to substitutealanine, asparagine or histidine for Gln11, a conserved residuethat donates a hydrogen bond to the reactive phosphoryl groupof bound substrate. Analysis of the binding and turnover ofnatural and synthetic substrates by the wild-type and mutantenzymes shows that the primary role of Gln11 is to prevent thenon-productive binding of substrate. 相似文献
10.
Wicker-Planquart C.; Canaan S.; Riviere M.; Dupuis L.; Verger R. 《Protein engineering, design & selection : PEDS》1996,9(12):1225-1232
Human gastric lipase (HGL) cDNA was synthesized by RT-PCR amplificationand cloned into the PVL 1392 baculovirus transfer vector. Therecombinant transfer vector was cotransfected with a modifiedbaculovirus DNA (BaculogoldTM) which contains a lethal deletion.Cotransfection of baculovirus DNA with the recombinant transfervector rescues the lethal deletion of this virus DNA and reconstitutesviable virus particles inside the transfected insect cells.BTI-TN-5B1-4 insect cells (also called High FiveTM cells) wereused to express recombinant HGL. The level of HGL secretionwas {small tilde}32 mg/1 of culture medium. The insect cellsalso accumulated HGL intracellularly, which indicated the existenceof rate-limiting steps in the secretion of HGL. Therefore weinvestigated the effect of replacing the HGL signal peptide(SP) by other SP of secreted proteins. The honeybee melittinSP and the human pancreatic lipase (HPL) SP were tested. Thefusion of HGL with HPL SP resulted in a 2-fold increase in theamount of lipase secreted from the insect cells. The recombinantactive HGL was not processed at the expected cleavage site ofthe natural enzyme, however, but at residue +3. On the otherhand, High FiveTM cells transfected with the vector encodingHGL fused to the melittin SP did not secrete any detectableactive HGL. Recombinant HGL was identified using the Westernblot procedure with rabbit polyclonal antibodies. The proteinmigrated with an apparent molecular mass of 45 kDa under SDSPAGEanalysis (compared with 50 kDa in the case of natural HGL),indicating that the insect cells have only a limited capacityto glycosylate HGL. The maximum specific activities of the recombinantlipase were 434, 730 and 562 units/mg using long-chain (IntralipidTM),medium-chain (trioctanoylglycerol) and short-chain (tributyroylglycerol)triacylglycerols, respectively. 相似文献
11.
Kruse R.; Hengstenberg W.; Beneicke W.; Kalbitzer H.R. 《Protein engineering, design & selection : PEDS》1993,6(4):417-423
The phosphocarrier HPr (heat stable protein) of Staphylococcuscarnosus was modified by site-directed mutagenesis of the correspondingptsH gene in order to analyse the importance of amino acidswhich were supposed to be part of the active centre of the protein.Three residues which are conserved in all HPrs, Argl7, Prol8and Glu84, were mutated: Argl7 was changed to His (17RH) andPro18 and Glu84 were changed into Ala (18PA and 84EA). In addition,Leu86 was changed into Ala (86LA) and one mutant protein wasmissing the last six residues of the HPr (83). The wild typegene and all mutant genes were overexpressed and the gene productspurified to homogeneity. Three-dimensional structures of wildtype and mutant proteins were monitored by NMR spectroscopy.All five mutant HPrs had native conformations. The ATP-dependentHPr kinase can phosphorylate all HPr derivatives at Ser46. ThePTS activity of the amino-terminal HPr mutant proteins 17RHand 18PA was different compared to wild type HPr. In contrast,the car boxy-terminal mutant HPrs possessed a similar enzymeactivity to the wild type HPr. The 17RH and 18PA HPrs with substitutionnear the active centre His15 showed a very slow phosphorylationby enzyme I but the further transfer of the phosphoryl groupto enzyme III was also strongly inhibited. The enzyme activityof the HPr 17RH was significantly improved at low pH. NMR pH-titrationexperiments showed that Arg17 is not responsible for the lowpKa, of the active centre His15 but this positively chargedresidue is essential in this position for the HPr activity. 相似文献
12.
Wu Zhen; Demma Mark; Strickland Corey L.; Syto Rosalinda; Le Hung V.; Windsor William T.; Weber Patricia C. 《Protein engineering, design & selection : PEDS》1999,12(4):341-348
Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of kcat; however, Km values for both peptide and FPPincreased 23-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity. 相似文献
13.
Vernet Thierry; Chatellier Jean; Tessier Daniel C.; Thomas David Y. 《Protein engineering, design & selection : PEDS》1993,6(2):213-219
A microbial expression system for the study of the cysteineprotease papain has been developed as a more useful alternativeto the insect cell/baculovirus expression system we have previouslyused. A synthetic papain precursor (propapain) gene was expressedin the yeast Saccharomyces cerevisiae under the control of the-factor promoter. Efficient expression required fusion of thepropapain sequence with the yeast -factor prepro region anda yeast host cell defective in the synthesis of vacuolar proteases.Surprisingly, the glycosylated form of the inactive papain precursoris not secreted, but accumulates within the yeast cell. Completeconversion of the intracellular zymogen into active mature papaincould be achieved in vitro. Purified recombinant papain producedby the yeast system has kinetic characteristics similar to thoseof the natural enzyme. An advantage of the yeast expressionsystem over the baculovirus/insect cell system is that we canperform mutagenesis and screening of papain mutants very efficiently.We have set up a one-tube screening procedurefor the simultaneous characterization of numerous mutants ofthe papain precursor. Yeast cells are grown and lysed in microtiterplate wells and the released papain precursor is then activatedto mature papain. This assay allows easy discrimination betweenproteins with close to wild type properties and proteins thatare not functional. We have applied this assay to investigatethe spectrum of amino acids which are tolerated at Asnl75 ofpapain using two independently derived libraries of mutantsat this position. Many amino acid substitutions at this positionare not accepted; only the reintroduction of Asn restored normalfunction. 相似文献
14.
Meijer Per-Johan; Holmberg Niklas; Grundstrom Gunilla; Bulow Leif 《Protein engineering, design & selection : PEDS》1996,9(11):1051-1054
Both freezing tolerance and NaCI tolerance are improved whenantifreeze proteins are expressed as fusion proteins with twodomains of staphylococcal protein A (SPA) in Escherichia coli.To characterize these properties further we created a randomlymutated expression library in E.coli, based on the winter flounderantifreeze protein HPLC-8 component gene. Low-fidelity PCR productsof this gene were fused to the spa gene encoding two domainsof the SPA. The library was screened for enhanced NaCl toleranceand four clones were selected. The freezing tolerance of eachof the selected clones was enhanced to varying extents. DNAsequencing of the isolated mutants revealed that the amphiphilicproperties of the native antifreeze protein were essentiallyconserved. Furthermore, by studying the primary sequence ofthe randomly mutated clones, in comparison with the degree offreezing tolerance, we have identified clues which help in understandingthe relationship between salt and freezing tolerance. 相似文献
15.
Thermolysin, a representative zinc metalloproteinase from Bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. The conventional expression method for recombinant thermolysin requires the autocatalytic cleavage, so that production of a mutant thermolysin is affected by its autocatalytic digestion activity. In this study, we have established a new expression method that does not require the autocatalytic cleavage. The mature sequence of thermolysin containing an NH(2)-terminal pelB leader sequence and the pre-prosequence of thermolysin were co-expressed constitutively in Escherichia coli as independent polypeptides under the original promoter sequences in the npr gene which encodes thermolysin. Unlike the conventional expression method, not only the wild-type thermolysin but also mutant thermolysins [E143A (Glu143 is replaced with Ala), N112A, N112D, N112E, N112H, N112K and N112R] were produced into the culture medium. The wild-type enzyme expressed in the present method was indistinguishable from that expressed in the conventional method based on autocatalytic cleavage, as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester. The present method should be useful especially for preparation of active-site mutants of thermolysin, which might have suppressed autocatalytic digestion activity. The results also demonstrate clearly that the covalent linking between the pro- and mature sequences is not necessary for the proper folding of the mature sequence by the propeptide in thermolysin. 相似文献
16.
White Christopher E.; Hunter Michael J.; Meininger David P.; White Lisa R.; Komives Elizabeth A. 《Protein engineering, design & selection : PEDS》1995,8(11):1177-1187
Fragments of human thrombomodulin (TM) have been expressed inlarge quantities in the Pichia pastoris yeast expression systemand purified to homogeneity. Fermentation of P.pastoris resultedin yields of 170 mg/1 TM. Purification to homogeneity resultedin an overall 10% yield, so that quantities of 20 mgpurified fragments can be readily obtained. Smaller fragmentsof TM, such as the individual fourth or fifth domains, werenot active, nor were equimolar mixtures of the two domains.These results demonstrate that the fourth and fifth epidermalgrowth factor (EGF)like domains together comprise thesmallest active fragment of TM. The fragment containing thefourth and fifth EGFlike domains (TMEGF(45)] had 10%the specific activity of rabbit TM. Comparison of the M388Lmutant TMEGF(45) fragment with the same mutant TMEGF(456)fragment showed that the fragment with the sixth domain hada 10fold better Km value for thrombin than the fragmentthat did not contain the sixth domain; this factor completelyaccounts for the higher specific activity of the fragments containingthe sixth domain. Comparison of the wildtype and M388Lmutants showed that the M388L mutation resulted in a 2foldincrease in kcat for the activation of protein C by the thrombinTMfragment complex, completely accounting for the 2foldincrease in specific activity of these mutant fragments. 相似文献
17.
Smith D.D.S.; Pratt K.A.; Sumner I.G.; Henneke C.M. 《Protein engineering, design & selection : PEDS》1995,8(1):13-20
A protein designed de novo to fold into the Greek key jellyrollstructural motif has been studied. Theoretical analyses haveindicated that the designed sequence should adopt the ß-strandarrangement of the Greek key jellyroll rather than any otherarrangement. A synthetic gene was constructed and the proteinexpressed in Escherichia coli. Circular dichroism spectroscopyis consistent with the protein folding into the designed conformationand also suggests the presence of tertiary structure. Fluorescencespectroscopy showed the single tryptophan to be partially buried,while denaturation studies showed changes in fluorescence toprecede alterations in secondary structure. 相似文献
18.
Mitaku Shigeki; Hirokawa Takatsugu 《Protein engineering, design & selection : PEDS》1999,12(11):953-957
The average hydrophobicity of a polypeptide segment is consideredto be the most important factor in the formation of transmembranehelices, and the partitioning of the most hydrophobic (MH) segmentinto the alternative nonpolar environment, a membrane or hydrophobiccore of a globular protein may determine the type of proteinproduced. In order to elucidate the importance of the MH segmentin determining which of the two types of protein results froma given amino acid sequence, we statistically studied the characteristicsof MH helices, longer than 19 residues in length, in 97 membraneproteins whose three-dimensional structure or topology is known,as well as 397 soluble proteins selected from the Protein DataBank. The average hydrophobicity of MH helices in membrane proteinshad a characteristic relationship with the length of the protein.All MH helices in membrane proteins that were longer than 500residues had a hydrophobicity greater than 1.75 (Kyte and Doolittlescale), while the MH helices in membrane proteins smaller than100 residues could be as hydrophilic as 0.1. The possibilityof developing a method to discriminate membrane proteins fromsoluble ones, based on the effect of size on the type of proteinproduced, is discussed. 相似文献
19.
Hewson Roger; Newbold Richard J.; Whitford David 《Protein engineering, design & selection : PEDS》1993,6(8):953-964
The DNA sequence of bovine microsomal cytochrome b5 has beenamplified from a liver cDNA library using a polymerase chainreaction. The amplified cDNA when cloned into plasmids thatsupport the high-level production of cytochrome bs in E.colileads to protein overexpression and results in cell coloniesbearing a strong red colouration. Using cassette mutagenesis,truncated versions of the cytochrome b5 cDNA have been madethat encode the first 90 amino acid residues (Ala1-Lys90), thefirst 104 amino acids (Ala1-Ser104) and the complete protein(Ala1-Asnl33). The location of the overexpressed cytochromebs within prokaryotic cells is dependent on the overall lengthof the protein. Expression of the Ala-Lys90 and Alal-SerlO4variants leads to a location in the cytoplasmic phase of thebacteria whereas the whole protein, Alal-Asnl33, is found withinthe bacterial membrane fraction. The last 30 residues of cytochromebs therefore contain all of the necessary information to insertthe protein into E.coli membranes. The solubility of the Alal-SerlO4variant permits the solution structure and stability of thisprotein to be measured using 1- and 2-D 1HNMR methods and electronicspectroscopy. 1-D NMR studies show that the chemical shiftsof the haem and haem ligand resonances of the Alal - Ser 104variant exhibit only very slight perturbations to their magneticmicroenvlronments when compared with the tryptic fragment offerricytochrome b5. These results indicate an arrangement ofresidues in the haem pocket that is very similar in both theAlal-Ser 104 variant and the tryptic fragment and by 2-D NMRit is shown that this similarity extends to the conformationsof the poly peptide backbone and side chains. Electronic spectroscopyof this variant shows absorbance maxima for the Soret peaksat 423 run (reduced) and 413 nm (oxidized). From absorbancespectra the relative thermal stabilities of the Alal-Ser 104variant and the tryptic fragment were measured. In the oxidizedstate the Ala1 - Ser104 variant denatures in a single cooperativetransition with a midpoint temperature (Tm of 73°C thatis significantly higher than that of tryptic ferricytochromebs. The reduced form of the protein shows increased transitiontemperatures (Tm 78°C) reflected in the values of Hm, Smand (G) of 420 kj/mol, 1096 J/mol/K and 12.38 kj/mol respectively,estimated for this variant. The increased stability of the Alal-SerlO4variant and other recombinant forms of cytochrome bs is correlatedwith the presence of additional residues at the N- and C-termini.The subtle differences in reactivity, stability and targetingbetween variant forms of cytochrome bs and the tryptic fragmentare discussed in terms of the overall structure of the protein. 相似文献
20.
Using the well-characterized antibody McPC603 as a model, wehad found that the Fv fragment can be isolated from Escherichiacoli as a functional protein in good yields, whereas the amountof the correctly folded Fab fragment of the same antibody producedunder identical conditions is significantly lower. In this paper,we analyse the reasons for this difference. We found that avariety of signal sequences function in the secretion of theisolated chains of the Fab fragment or in the co-secretion ofboth chains in E.coli. The low yield of functional Fab fragmentis not caused by inefficient expression or secretion in E.coli,but by inefficient folding and/or assembly in the periplasm.We compared the folding yields for the Fv and the Fab fragmentin the periplasm under various conditions. Several diagnosticframework variants were constructed and their folding yieldsmeasured. The results show that substitutions affecting cis-prolineresidues and those affecting various disulphide bonds in theprotein are by themselves insufficient to dramatically changethe partitioning of the folding pathway to the native structure,and the cause must lie in a facile aggregation of folding intermediatescommon to all structural variants. However, all structural variantscould be obtained in native form, demonstrating the generalutility of the secretory expression strategy. 相似文献