Insertion of a disulfide-containing neurotoxin into E.coil alkaline phosphatase: the hybrid retains both biological activities

We have inserted a disulfide-containing snake neurotoxin intothe N-terminal end of Escherichia coli alkaline phosphatase,between residues +6 and +7 of the mature enzyme. For this purpose,we have designed a cloning and expression vector which allowsinsertion of foreign DNA between the corresponding codons, andvisual selection of the desired recombinant clones upon recoveryof phosphatase activity. The hybrid protein is exported to thebacterial periplasm, the alkaline phosphatase signal peptideis correctly processed, and both domains are functionally conformed.The phosphatase domain displays catalytic activity, and theinserted toxin is able to bind to its biological target, thenicotinic acetylcholine receptor. The hybrid molecule is remarkablystable and resistant to proteolysis. Crude periplasmic extractcontaining the hybrid can be used as a tracer-containing reagentin competitive enzymo-immuno and enzymo-receptor assays. Wepropose to use the system described in this paper for fast preparationof properly folded disulfide-containing enzymatic probes.     

Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase

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     

Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein--single-chain antibody fusion protein

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, 837–841]. 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.     

High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling

Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.     

The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli

In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from V_H manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation.     

Studies on chimeric fusion proteins of human aldolase isozymes A and B

Several kinds of fusion proteins between human aldolases A andB were prepared by recombinant DNA technology and their enzymicproperties were examined. AB chimeras, which have aldolase Aat the N-terminal region and aldolase B at the C-terminal region,were scarcely obtained, while BA chimeras were abundant (Kitajimaet al., (1990), J. Biol. Chem., 265, 17493-17498). All the BABchimeras, aldolase A fragments inserted in aldolase B, showedactivity assignable to aldolase B type, which imply an essentialrole of Tyr residue at the C-terminus of aldolase A in the bindingof fructose-l,6-bisphosphate (Fni-1,6-P₂). BAB chimeras alsoshowed reactivity to effectors such as fructose-2,6-bisphosphate(Fru-2,6-P₂) and pyridoxal 5-phosphate (PLP), in a similar mannerto aldolase B. BAB108 has a similarity to the BA108 chimera,but acts differently from other BAB chimeras, suggesting thatits structure around active site looks like that of aldolaseA     

A method for construction of long randomized open reading frames and polypeptides

A method is presented for construction of randomized open readingframe sequences (ORFs) and gene libraries containing them. Thebuilding blocks for the ORFs were 75 bp long DNA fragments generatedby cloning sequences from a single synthetic oligonucleotidepreparation by bridge mutagenesis. The fragments had the propertythat, regardless of their orientation in the ligated product,the ORF of the construct was maintained. The heterogeneity ofthe ORFs resulted from the random ligation of 2000 differentDNA fragments. The randomized ORFs were cloned downstream fromthe lac promoter in a multicopy plasmid in Escherichia coli.To test the method, a library of 10⁶ clones was constructed.     

Recombinant rabbit uteroglobin expressed at high levels in E.coli forms stable dimers and binds progesterone

In order to express uteroglobin in Escherichia coli we haveconstructed a DNA coding for complete mature rabbit uteroglobinby fusing genomic sequences from the second exon of the geneto an incomplete cDNA. This DNA was inserted into various positionsof the polylinker cloning region of pDS expression vectors andthe uteroglobin gene was expressed in E.coli by IPTG induction.Four different uteroglobinderived proteins were produced containing1, 3,5 and 7 more N-terminal amino acids than the naturallyoccurring mature protein. The yield of soluble protein stronglyincreased with increasing length of the N-terminal additions.Protein and RNA analysis showed that this variation is mostlikely due to progressively higher translation efficienciesof the larger recombinants. UG7, the most efficiently synthesizedrecombinant protein, carrying seven additional N-terminal aminoacids, was purified and further characterized. Like naturaluteroglobin, UG7 forms a dimer and binds progesterone with anaffinity indistinguishable to the natural protein. This bacteriallyproduced protein can be used for detailed structure–functioninvestigations of uteroglobin.     

Protien side-chain conformational entropy derived from fusion data-comparison with other empirical scales

The loss of conformational entropy of protein side-chains isa major effect in the energetics of folding. The simplest approachis to enumerate the number of freely rotatable bonds. Recently,two scales of side-chain conformational entropy have been proposedbased on the definition of entropy as the Boltzmann samplingover all accessible states (S –Rp_iInp_i, where p_i is theprobability of being in a rotameric state). In one scale, derivedonly for aliphatic and aromatic side-chains, the values of p_iwere obtained from Monte Carlo simulations. In the other scale,the observed frequencies of different rotameric states in adatabase of protein crystal structures yielded an estimate forp_i. Here an empirical estimation of the fusion entropy of theside-chains is used to derive a third scale. The fusion entropyis obtained as a sum of empirically derived contributions fromcomponent hydrocarbon and functional groups. There is a goodagreement between the fusion scale and the other two scales.This suggests that the magnitude of conformational entropy isbeing correctly established     

A combinatorial library of an {alpha}-helical bacterial receptor domain

The construction and characterization of a combinatorial libraryof a solvent-exposed surface of an -helical domain derived froma bacterial receptor is described. Using a novel solid-phaseapproach, the library was assembled in a directed and successivemanner utilizing single-stranded oligonucleotides containingmultiple random substitutions for the variegated segments ofthe gene fragment The simultaneous substitution of 13 residuesto all 20 possible amino acids was carried out in a region spanning81 nucleotides. The randomization was made in codons for aminoacids that were modelled to be solvent accessible at a surfacemade up from two of the three a-helices of a monovalent Fc-bindingdomain of staphylococcal protein A. After cloning of the PCR-amplifiedlibrary into a phagemid vector adapted for phage display ofthe mutants, DNA sequencing analysis suggested a random distributionof codons in the mutagenized positions. Four members of thelibrary with multiple substitutions were produced in Escherichiacoli as fusions to an albumin-binding affinity tag derived fromstreptococcal protein G. The fusion proteins were purified byhuman serum albumin affinity chromatography and subsequentlycharacterized by SDSelectrophoresis, CD spectroscopy and biosensoranalysis. The analyses showed that the mutant protein A derivativescould all be secreted as soluble full-length proteins. Furthermore,the CD analysis showed that all mutants, except one with a prolineintroduced into helix 2, have secondary structures in closeagreement with the wild-type domain. These results proved thatmembers of this -helical receptor library with multiple substitutionsin the solvent-exposed surface remain stable and soluble inE.coli. The possibility of using this library for a phenotypicselection strategy to obtain artificial antibodies with novelfunctions is discussed.     

The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides

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 46–66 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.     

Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli

We have developed a high-level production system for the C-terminaldomain of secretory leukoprotease inhibitor (SLPI) to investigateits pharmacological activities. A gene for the C-terminal domainof SLPI, (Asn55-AlalO7)SLPI, was constructed from chemicallysynthesized deoxyoligonucleotides. It was fused to a gene forthe N-terminal portion of human growth hormone via a DNA sequenceencoding Leu-Val-Pro-Arg, which can he cleaved by thrombin.The fused gene was expressed in Escherichia coli under the controlof a trp promoter, and the fusion protein was obtained as aninclusion body. After sulfonation of the cysteine residues,the sulfonated fusion protein was cleaved at the desired siteby thrombin. Sulfonated (Asn55-Ala107)SLPI was refolded in Trisbuffer containing reduced and oxidized glutathione. The resulting(Asn55-Ala107) SLPI was purified by cation-exchange chromatographyand reverse-phase high performance liquid chromatography. Thefinal yield was 50 mg/l culture. (Asn55-Ala107)SLPI was as activeagainst elastase as, but had less trypsin inhibitory activitythan, native SLPI. This system is suitable for the large-scaleproduction of the C-terminal domain of SLPI, which is an elastase-specificinhibitor.     

Expression, purification and characterization of recombinant crambin

Crambin, a small hydrophobic protein (4.7 kDa and 46 residues),has been successfully expressed in Escherichia coli from anartificial, synthetic gene. Several expression systems wereinvestigated. Ultimately, crambin was successfully expressedas a fusion protein with the maltose binding protein, whichwas purified by affinity chromatography. Crambin expressed asa C-terminal domain was then cleaved from the fusion proteinwith Factor Xa protease and purified. Circular dichroism spectroscopyand amino acid analysis suggested that the purified materialwas identical to crambin isolated from seed. For positive identificationthe protein was crystallized from an ethanol–water solution,by a novel method involving the inclusion of phospholipids inthe crystallization buffer, and then subjected to crystallographicanalysis, Diffraction data were collected at the Brookhavensynchrotron (beamline-X12C) to a resolution of 1.32 Åat 150 K. The structure, refined to an R value of 9.6%, confirmedthat the cloned protein was crambin. The availability of clonedcrambin will allow site-specific mutagenesis studies to be performedon the protein known to the highest resolution.     

Engineered turns of a recombinant antibody improve its in vivo folding

Using recombinant antibodies functionally expressed by secretionto the periplasm in Escherichia coli as a model system, we identifiedmutations located in turns of the protein which reduce the formationof aggregates during in vivo folding or which influence cellstability during expression. Unexpectedly, the two effects arebased on different mutations and could be separated, but bothmutations act synergistically in vivo. Neither mutation increasesthe thermodynamic stability in vitro. However, the in vivo foldingmutation correlates with the yield of oxidative folding in vitro,which is limited by the side reaction of aggregation. The invivo folding data also correlate with the rate and activationentropy of thermally induced aggregation. This analysis showsthat it is possible to engineer improved frameworks for semi-syntheticantibody libraries which may be important in maintaining librarydiversity. Moreover, limitations in recombinant protein expressioncan be overcome by single amino acid substitutions.     

Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO)contains three disulphide bridges linking residues C⁴¹XXC⁴⁴,C¹⁵⁵XXC¹⁵⁸, C¹⁷³XXXXC¹⁷⁸. To get information on the role playedby these cross-links in determining the structural and functionalproperties of the protein, we performed site-directed mutagenesison Cys residues and investigated the changes in folding, stabilityand functional features of the mutants and analysed the resultswith computational analysis. The reductase activity of SsPDOand its mutants was evaluated by insulin and thioredoxin reductaseassays also coupled with peroxiredoxin Bcp1 of S. solfataricus.The three-dimensional model of SsPDO was constructed and correlatedwith circular dichroism data and functional results. Biochemicalanalysis indicated a key function for the redox site constitutedby Cys155 and Cys158. To discriminate between the role of thetwo cysteine residues, each cysteine was mutagenised and thebehaviour of the single mutants was investigated elucidatingthe basis of the electron-shuffling mechanism for SsPDO. Finally,cysteine pK values were calculated and the accessible surfacefor the cysteine side chains in the reduced form was measured,showing higher reactivity and solvent exposure for Cys155.     

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression

Site-directed mutagenesis of the Lactococcus lactis lacR genewas performed to identify residues in the LacR repressor thatare involved in the induction of lacABCDFEGX operon expressionby tagatose-6-phosphate. A putative inducer binding domain locatednear the C-terminus was previously postulated based on homologystudies with the Escherichia coli DeoR family of repressors,which all have a phosphorylated sugar as inducer. Residues withinthis domain and lysine residues that are charge conserved inthe DeoR family were changed into alanine or arginine. The productionof the LacR mutants K72A, K80A, K80R, D210A, K213A and K213Rin the LacR-deficient L.lactis strain NZ3015 resulted in repressedphospho-ß-galactosidase (LacG) activities and decreasedgrowth rates on lactose. Gel mobility shift assays showed thatthe complex between a DNA fragment carrying the lac operatorsand LacR mutants K72A, K80A, K213A and D210A did not dissociatein the presence of tagatose-6-phosphate, in contrast to wildtype LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A,F214R, R216R and R216K) exhibited no gross effects on inducerresponse. The results strongly suggest that the lysines at positions72, 80 and 213 and aspartic acid at position 210 are involvedin the induction of lac operon expression by tagatose-6-phosphate.     

Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus

The crystal structure of a serine protease from the alkalophilicstrain Bacillus alcalophilus PB92 has been determined by X-raydiffraction at 1.75 Â resolution. The structure has beensolved by molecular replacement using the atomic model of subtilisinCarlsberg. The model of the PB92 protease has been refined toan R-factor of 14.0% and contains 1882 protein atoms, two calciumions and 188 water molecules. The overall folding of the polypeptide chain closely resembles that of the subtilisins. Furthermore,almost all of the secondary structure elements found in subtilisinCarlsberg are also present in the PB92 protease. The major differencesbetween the two structures are located around the deletion regions(residues 37 and 158–161 in subtilisin Carlsberg) andin two loops which are known to be the most variable parts ofsubtilisin structures. Flexibility of one of these loops (residues126–130 in the PB92 protease) is believed to account forthe inducedfit mechanism of substrate binding.     

Potential of genetic algorithms in protein folding and protein engineering simulations

Genetic algorithms are very efficient search mechanisms whichmutate, recombine and select amongst tentative solutions toa problem until a near optimal one is achieved. We introducethem as a new tool to study proteins. The identification andmotivation for different fitness functions is discussed. Theevolution of the zinc finger sequence motif from a random startis modelled. User specified changes of the repressor structurewere simulated and critical sites and exchanges for mutagenesisidentified. Vast conformational spaces are efficiently searchedas illustrated by the ab initio folding of a model protein ofa four ß strand bundle. The genetic algorithm simulationwhich mimicked important folding constraints as overall hydrophobicpackaging and a propensity of the betaphilic residues for transpositions achieved a unique fold. Cooperativity in the ßstrand regions and a length of 3–5 for the interconnectingloops was critical. Specific interaction sites were considerablyless effective in driving the fold.