Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli

An advantage of exporting a recombinant protein to the periplasmof Escherichia coli is decreased proteolysis in the periplasmcompared with that in the cytoplasm. However, protein degradationin the periplasm also occurs. It has been widely accepted thatthe thermodynamic stability of a protein is an important factorfor protein degradation in the cytoplasm of E.coli. To investigatethe effect of the thermodynamic stability of an exported proteinon the extent of proteolysis in the periplasm, barnase (an extracellularribonuclease from Bacillus amyloliquefaciens) fused to alkalinephosphatase leader peptide was used as a model protein. A setof singly or doubly mutated barnase variants were constructedfor export to the E.coli periplasm. It was found that the half-lifeof the barnase variants in vivo increased with their thermodynamicstability in vitro. A dominant factor for the final yield ofexported barnase was not exportability but the turnover rateof the barnase variant. The yield of a stabilized mutant wasup to 50% higher than that of the wild type. This suggests thatexporting a protein to the periplasm and using protein engineeringto enhance the stability can be combined as a strategy to optimizethe production of recombinant proteins.     

Conformation and stability of barley chymotrypsin inhibitor-2 (CI-2) mutants containing multiple lysine substitutions

A major goal of agricultural biotechnology is to increase thenutritional value of maize seed through the expression of heterologousproteins enriched in lysine. One promising candidate is barleychymotrypsin inhibitor-2 (CI-2), a plant protein that has beenextensively characterized with respect to structure and function.Based on the tertiary structure of wild-type (WT) CI-2, fivemutants with lysine contents ranging from 20 to 25 mol percentwere designed, expressed in Escherichia coli and purified byion exchange and gel permeation chromatography. Inasmuch asprevious transgenic experiments suggested that proper foldingand stability may be essential for in vivo accumulation of theengineered proteins in plant cells, we first undertook an invitro study of the conformation and thermodynamic stabilityof the CI-2 mutants in order to select an ideal candidate forplant expression. Mutant and WT CI-2 proteins had similar circulardichroism spectra, suggesting similar secondary structures.However, differences in the accessibility of the sole tryptophanresidue, Trp24, indicated that the local conformation differedamong the mutants. The thermodynamic stability of the mutantsranged from <2 to 4.9 kcal/mol compared with ~7 kcal/molfor the wild-type protein. In conjunction with proteolytic stabilitystudies, we have identified one mutant that has the potentialto be expressed in a stable manner in plant cells.     

Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence

Statistical analysis of 12 unstable and 32 stable proteins revealedthat there are certain dipeptides, the occurrence of which issignificantly different in the unstable proteins compared withthose in the stable ones. Based on the impact of these dipeptideson the unstable proteins over the stable ones, a weight valueof instability is assigned to each of the dipeptides. For agiven protein the summation of these weight values normalizedto the length of its sequence helps to distinguish between unstableand stable proteins. Results suggest that the in vivo instabilityof proteins is possibly determined by the order of certain aminoacids in its sequence. An attempt is made to correlate metabolicstability of proteins with features of their primary sequencewhere weight values of instability for a protein of known sequencecould thus be used as an index for predicting its stabilitycharacteristics.     

Alignment of protein sequences using secondary structure: a modified dynamic programming method

A method for comparison of protein sequences based on theirprimary and secondary structure is described. Protein sequencesare annotated with predicted secondary structures (using a modifiedChou and Fasman method). Two lettered code sequences are generated(Xx, where X is the amino acid and x is its annotated secondarystructure). Sequences are compared with a dynamic programmingmethod (STRALIGN) that includes a similarity matrix for boththe amino acids and secondary structures. The similarity valuefor each paired two-lettered code is a linear combination ofsimilarity values for the paired amino acids and their annotatedsecondary structures. The method has been applied to eight globinproteins (28 pairs) for which the X-ray structure is known.For protein pairs with high primary sequence similarity (>45%),STRALIGN alignment is identical to that obtained by a dynamicprogramming method using only primary sequence information.However, alignment of protein pairs with lower primary sequencesimilarity improves significantly with the addition of secondarystructure annotation. Alignment of the pair with the least primarysequence similarity of 16% was improved from 0 to 37% ‘correct’alignment using this method. In addition, STRALIGN was successfullyapplied to seven pairs of distantly related cytochrome c proteins,and three pairs of distantly related picornavirus proteins.     

Crystallographic phases through genetic engineering: experiences with colicin A

Colicins are antibiotic proteins that kill sensitive Escherichiacoli cells. The structure of the pore-forming fragment of colicinA has been solved to 2.5 A resolution using the techniques ofX-ray crystallography and genetic engineering. Site-directedmutagenesis was used to construct a number of cysteine-containingmutant proteins, one of which yielded an excellent mercurialderivative. Our experiences suggest strategies for obtaininguseful heavy-atom derivatives for protein crystallography usinggenetic engineering techniques.     

Cellulose-binding domains: potential for purification of complex proteins

The endoglucanase CenA and the exoglucanase Cex from Cellulomonasfimi each contain a discrete cellulose-binding domain (CBD),at the amino-terminus or carboxyl-terminus respectively. Thegene fragment encoding the CBD can be fused to the gene of aprotein of interest. Using this approach hybrid proteins canbe engineered which bind reversibly to cellulose and exhibitthe biological activity of the protein partner. Alkaline phosphatase(PhoA) from Escherichia coli, and a ß-glucosidase(Abg) from an Agrobacterium sp. are dimeric proteins. The fusionpolypeptides CenA-PhoA and Abg-CBC_cex are sensitive to proteolysisat the junctions between the fusion partners. Proteolysis resultsin a mixture of homo- and heterodimers; these bind to celluloseif one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoAand CenA-PhoA/PhoA. CBD fusion polypeptides could be used inthis way to purify polypeptides which associate with the fusionpartner.     

Monte Carlo docking of protein-DNA complexes: incorporation of DNA flexibility and experimental data

A Monte Carlo simulation program (MONTY) has been developedto dock proteins onto DNA. Protein and DNA interact via square-wellpotentials for hydrogen bond and van der Waals interactions.The effect of the inclusion of DNA flexibility and experimentallyderived restraints has been tested on members of the helix-turn-helixfamily of DNA binding proteins. Unwinding and bending the DNAdouble helix improves the number of correctly retrieved hydrogenbonds in simulations starting from the 434 cro protein monomercomplexed with a standard B-DNA O_Rl half-site. Agreement withphosphate ethylation interference and mutagenesis data is rewardedwith energy bonuses. This protocol was tested on protein-DNAcomplexes of 434 cro, lac headpiece and a mutant lac headpieceresembling the gal repressor headpiece with the recognitionhelices in correct and reversed orientations in the DNA majorgroove. The inclusion of experimental data gives an improvedconvergence of the correctly oriented structures and allowsfor an easier discrimination between correctly and incorrectlydocked complexes     

Coordinated amino acid changes in homologous protein families

In the tobamovirus coat protein family, amino acid residuesat some spatially close positions are found to be substitutedin a coordinated manner [Altschuh et al. (1987) J. Mol. Biol.,193,693]. Therefore, these positions show an identical patternof amino acid substitutions when amino acid sequences of thesehomologous proteins are aligned. Based on this principle, coordinatedsubstitutions have been searched for in three additional proteinfamilies: serine proteases, cysteine proteases and the haemoglobins.Coordinated changes have been found in all three protein familiesmostly within structurally constrained regions. This methodworks with a varying degree of success depending on the functionof the proteins, the range of sequence similarities and thenumber of sequences considered. By relaxing the criteria forresidue selection, the method was adapted to cover a broaderrange of protein families and to study regions of the proteinshaving weaker structural constraints. The information derivedby these methods provides a general guide for engineering ofa large variety of proteins to analyse structure–functionrelationships.     

Replacement of cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c with threonine: improved stability of the mutant protein

Site-directed mutagenesis has been used to change the codonfor cysteine-107 of Saccharomyces cerevisiae iso-1-cytochromec to a threonine codon. The resulting protein is active in vivo,is methylated as in the wild-type protein and has optical propertiesindistinguishable from those of the wild-type protein. The threonine-107iso-1-cytochrome c demonstrated fully reversible electrochemicalbehaviour and a mid-point reduction potential of 272 mV versusNHE. In addition, this mutant does not demonstrate a tendencyto autoreduce or to dimerize as does the wild-type protein.These properties of the threonine-107 mutant establish thatit will provide a useful background in which to make subsequentmutations for mechanistic and physical studies of yeast iso-1-cytochromec.     

The role of heat-shock and chaperone proteins in protein folding: possible molecular mechanisms

Recently some heat-shock proteins have been linked to functionsof ‘chaperoning’ protein folding in vivo. Here currentexperimental evidence is reviewed and possible requirementsfor such an activity are discussed. It is proposed that onemode of chaperone action is to actively unfold misfolded orbadly aggregated proteins to a conformation from whkh they couldrefold spontaneously; that improperly folded proteins are recognizedby excessive stretches of solvent-exposed backbone, rather thanby exposed hydrophobic patches; and that the molecular mechanismfor unfolding is either repeated binding and dissociation (‘plucking’)or translocation of the protein backbone through a binding cleft(‘threading’), allowing the threaded chain to refoldspontaneously. The observed hydrolysis of ATP would providethe energy for active unfolding. These hypotheses can be appliedto both monomeric folding and oligomeric assembly and are sufficientlydetailed to be open to directed experimental verification.     

Targeting myeloid leukemia with a DT390-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice

The IL-3 receptor was expressed on a high frequency of myeloidleukemia cells and also on hematopoietic and vascular cells.We previously showed that a recombinant IL-3 fusion immunotoxin(DT₃₉₀IL-3) expressed by splicing the murine IL-3 gene to atruncated diphtheria toxin (DT₃₉₀) gene selectively killed IL-3R⁺expressing cells and was not uniformly toxic to uncommited BMprogenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J.and Vallera,D.A., 1996, Blood, 88, 1445–1456). Thus, weexplored the feasability of using DT₃₉₀IL-3 as an anti-leukemiaagent. DT₃₉₀IL-3 was toxic when administered to mice at dosesas low as 0.1 µg/day. The dose limiting toxicity appearedto be related to platelet and bleeding effects of the fusiontoxin. Because of these effects, DT₃₉₀IL-3 was studied ex vivoas a means of purging contaminating leukemia cells from BM graftsin a murine autologous BM transplantation. In this setting,as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid32Dp210 leukemia cells were lethal. An optimal purging intervalof 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BMmixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneicmice. Mice given treated grafts containing BM and a lethal doseof 32Dp210 cells survived over 100 days while mice given untreatedgrafts did not survive (P < 0.00001). DT₃₉₀IL-3 may provehighly useful for ex vivo purging of lethal malignant leukemiacells from autologous BM grafts.     

Engineering a de novo-designed coiled-coil heterodimerization domain for the rapid detection, purification and characterization of recombinantly expressed peptides and proteins

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 peptide–protein or peptide–ligand interactions.Therefore this system offers a new alternative to existing peptideor protein fusion tags and demonstrates the utility of a denovo-designed system.     

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.     

An approach to protein homology modelling based on an ensemble of NMR structures: application to the Sox-5 HMG-box protein

A new approach has been developed to reduce multiple proteinstructures obtained from NMR structure analysis to a smallernumber of representative structures which still reflect thestructural diversity of the data sets. The method, based onthe clustering of similar structures, has been tested in thehomology model building of the structure of Sox-5, a sequence-specificDNA-binding protein belonging to the high mobility group (HMG)nuclear proteins family. Sox (SRY box) genes are the autosomalgenes related to the sex-determining SRY, Y chromosomal gene.The Sox-5 protein, encoded by one of the SRY-related genes,displays a 29% sequence identity with the HMG1 B-box domainwhose structure, determined previously by NMR, has been usedin our study to predict the structure of Sox-5. Two independentensembles of HMG1 structures, each represented by closely relatedcoordinate sets, were used. Nine representative structures forHMG1 were subsequently selected as starting points for the modellingof Sox-5. The model of the protein shows close similarity tothe HMG1 fold, with differences at the secondary structure levellocated mainly in a-helices 1 and 3. A left-handed, three residueper turn polyproline II helix, forming a conserved polyprolineII/-helix supersecondary motif, was identified in the N-terminalregion of Sox-5 and other HMG boxes.     

Mutational analysis supports a structural model for the cell cycle protein kinase p34

Structural models for the eukaryotic cell cycle control proteinp34 from human, S.pombe and S.cerevisiae have been derived fromthe crystallographic coordinates of the cAMP-dependent proteinkinase (cAPK) catalytic subunit (active conformation) and comparedwith the structure of Inactive CDK2 apoenzyme. Differences betweenthe p34 and cAPK catalytic sites provide a possible explanationfor their different substrate specificities. The p34 modelslocalize Tyrl5 and Thrl4 close to the sites of catalysis andsubstrate recognition where their phosphorylatlon could inhibitp34 kinase activity either by blocking MgATP or substrate binding.The conserved sequences PSTAIRE and LYLIFEFL are both closeto the catalytic site and accessible on the protein surfaceavailable to mediate interactions with other proteins. It ispredicted that p34 has an active-site cleft composed almostentirely of sequences common to all protein kinases and sequencesunique to the p34 protein family. Genetic and biochemical analysesof p34 have shown that it interacts extensively with a numberof other proteins. The model allows the relative dispositionof these sites of mutation to each other and to the sites ofcatalysis and substrate recognition to be appreciated. Surfaceregions on p34 that are important for function have been identified.These sites identify residues that may interact with p13^SUCL,cydin, plO7^WEEL and p80^cdc25     

Genetically engineered brain drug delivery vectors: cloning, expression and in vivo application of an anti-transferrin receptor single chain antibodystreptavidin fusion gene and protein

A single chain Fv antibody–streptavidin 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 blood–brain 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 antibody–streptavidin 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 biotin–polyethyleneglycol–fluorescein to theOX26 single chain Fv antibody–streptavidin fusion proteinresulted in illumination of isolated rat brain capillaries inconfocal fluorescent microscopy. In conclusion, these studiesdemonstrate that genetically engineered single chain Fv antibody–streptavidinfusion proteins may be used for non-invasive neurotherapeuticdelivery to the brain using endogenous BBB transport systemssuch as the transferrin receptor.     

A pore-forming protein with a metal-actuated switch

Staphylococcal -hemolysin, a pore-forming exotoxin, is a polypeptideof 293 amino acids that is secreted by Staphylococcus aureusas a water-soluble monomer. It assembles to form hexameric poresin lipid bilayers. Previous studies of pore formation have establishedthe involvement of a central glycine-rich loop. Here, we showthat when five consecutive histidine residues replace aminoacids 130–134 at the midpoint of the loop, they providea switch with which pore activity can be (i) turned off by micromolarconcentrations of divalent zinc ions and (ii) turned back onwith the chelating agent EDTA. Planar bilayer recordings showthat Zn²⁺ and EDTA can act on open channels from either sideof the bilayer and thus demonstrate that the central loop linespart of the conductive pathway. Our results suggest that genetically-engineeredpore-forming proteins might make useful components of metalion sensors     

Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli

Bovine somatotropin (bST) was secreted from Escherichia coliat moderate levels of 1–2 µg/ml/OD using expressionvectors in which the bST gene was fused to the lamB secretionsignal. To study the secretion properties of bST in E.coli further,two approaches for modifying the secretion signal were employed.In the first case, fusion proteins were constructed with sixalternative bacterial secretion signals: three from E.coli proteins(HisJ, MalE and OmpA), two from bacteriophage proteins (M13coat protein and PA-2 Lc) and one from the chitinase A proteinof Serratia marcescens. The results, as monitored by Westernblot analysis of both total cell protein and the periplasmicfraction, showed that these changes in the secretion signaldid not significantly affect the secretion properties of bST.In the second approach, a library of random mutations was createdin the lamB secretion signal and 200 independent clones werescreened. The level of secreted bST was determined by growingindividual clones in duplicate in microtiter wells, inducingprotein expression and measuring the bST released by osmoticshock using a particle concentration fluorescent immunoassay.The secretion properties of several novel variants in the LamBsignal peptide are presented.     

The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis

The Escherichia coli aspartate receptor is a dimer with twotransmembrane sequences per monomer that connect a periplasmicligand binding domain to a cytoplasmic signaling domain. Themethod of 'hydrophobic-biased' random mutagenesis, that we describehere, was used to construct mutant aspartate receptors in whicheither the entire transmembrane sequence or seven residues nearthe center of the transmembrane sequence were replaced withhydrophobic and polar random residues. Some of these receptorsresponded to aspartate in an in vivo chemotaxis assay, whileothers did not. The acceptable substitutions included hydrophobicto polar residues, small to larger residues, and large to smallerresidues. However, one mutant receptor that had only a few hydrophobicsubstitutions did not respond to aspartate. These results addto our understanding of sequence specificity in the transmembraneregions of proteins with more than one transmembrane sequence.This work also demonstrates a method of constructing familiesof mutant proteins containing random residues with chosen characteristics.     

Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system

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, ETA^1–413.DHFR^1–187.ETA^609–613.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.