Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli

Bacterial expression systems can greatly facilitate proteinengineering of antibodies. We have developed a system for high-levelexpression of antibodies, antibody fragments, or hybrid antibodieswith novel effector functions in the periplasm of Escherichiacoli. From 5 ml of cells, a simple extraction yields sufficientmaterial for SDS-gel electro-phoresis, detection and characterizationof hapten binding. To demonstrate our system, heavy-chain variableregions and 1 light chains of a mouse anti-NP antibody weresynthesized as hybrid proteins with a bacterial signal peptide(Omp F). Each chain is secreted into the periplasm where processing(cleavage of the signal peptide), folding and heterodimer associationtake place. Periplasmic proteins are released by cold osmoticshock, and hapten-binding activity is easily detected withoutfurther manipulation. The ease of genetic engineering in thissystem will facilitate the production of immunoglobulin derivativesdesigned for specific applications, and expression of thesemolecules in a native state will allow the rapid screening ofcombinatorial libraries and the results of mutagenesis.     

Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine

The Fab region of an IgG2b antibody (AM7B2.1) reactive to theherbicide atrazine was cloned into a plasmid vector using thepolymerase chain reaction and two sets of degenerate oligonucleotideprimers designed to mimic the amino acid variation at the N-terminiof _L-chains and TH-chains. These primers also provide a secretionsignal fused precisely to the antibody gene sequence for secretionof the mature antibody. A further set of universal oligonucleotideprimers was developed for the direct sequencing of the V_H andC_m regions of _B-chains and the V_L and C_L regions of _L-chainswithout subcloning and were used to determine the sequence ofthis antibody. The _L-chain was found to not possess a conservedCys residue at position 23 and the implications of this observationare discussed. The cloned genes were expressed in Escherichiacoli using a commercially available T7 RNA polymerase-basedplasmid. The clones were also expressed in a 17 RNA polymerasebasedsystem containing an attenuated version of the T7 RNA polymerasepromoter, plus a lac promoter placed in an antisense orientation,to enhance plasmid stability. The expressed products were confirmedas atrazine reactive by binding to an atrazine derivative conjugatedwith alkaline phosphatase.     

Sequence requirements for cytochromes P450IIA1 and P450IIA2 catalytic activity: evidence for both specific and non-specific substrate binding interactions through use of chimeric cDNAs and cDNA expression

Cytochrome P450s IIA1 and IIA2, encoded by the CYP2A1 and CYP2A2genes, display 88% amino acid sequence similarities. The dissimilaritiesof sequence between these two enzymes are primarily localizedwithin four discrete regions of the polypeptides that are separatedby regions of absolute sequence identity. IIA1 specificallyhydroxylates the prototype substrate testosterone at the 7 and6 position with a predominance of 7 metabolite. IIA2, on theother hand, hydroxylates this steroid at eight positions onthe molecule, with one of the most abundant metabolites being15hydroxytestosterone. To determine those amino acids responsiblefor the difference in testosterone hydroxylation specificities,chimeras were constructed between IIA1 and IIA2 cDNAs and expressedin cell culture using vaccinia-virus-mediated cDNA expression.Chimeras, in which the first 355 amino acids correspond to asingle enzyme, maintain the specificity associated with thatenzyme. Of six chimeras which have substitutions between aminoacids 161 and 276, two are inactive and the remaining four givesimilar metabolite profiles, in which both 7 and 15 hydroxylationspecificities have been lost. Two of these four chimeras arediametric apposites, suggesting that modification of eitherthe N-terminal or central regions of the enzymes results inconformational changes that prevent the specific binding interactionsresponsible for the narrow regioselectivity associated withIIA1 and 15-hydroxytestosterone formation associated with IIA2.     

Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production: application to an antibody fragment

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.     

The construction and cloning of synthetic genes coding for artificial proteins and expression studies to obtain fusion proteins

Synthetic genes coding for artificial proteins with predeflnedand nutritionally valuable amino acid compositions have beenconstructed and cloned In bacterial plasmid vector pKK233-2.The genes were constructed from three easily interchangeable‘cassettes’ encoding either essential, non-essentialor branched-chain amino acid residues. A potential hairpin loopstructure in the mRNA around the region of the ribosome bindingsite was probably the reason for blockage of translation fromthis vector. Two selected genes, AHB (containing one copy ofeach cassette) and A (consisting of six copies concatemerizedA₆cassette) were cloned into pUR300, a (ß-Gal fusionvector and expressed as fusion proteins (ß-Gal-AHBand (ß-Gal-A₆.     

Expression of an exogenous peptide epitope genetically engineered in the variable domain of an immunoglobulin: implications for antibody and peptide folding

Immunoglobulins bind antigens and express individual antigenicspecificities mainly through residues located in hypervariableloops of their N-terminal domains. Hyper-variable loops arekept in place by a molecular scaffold organized in a sandwich-likestructure with two ß-sheets stabilized by a disulfidebridge (the immunoglobulin fold). This structural feature, togetherwith the possibility of obtaining high level expression, extracellularsecretion, easy purification and stability of the protein product,render immunoglobulin an ideal ‘molecular vehicle’for the expression of exogenous peptides. Here we report onthe engineering of an immunoglobulin expressing an exogenousepitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP)₃.By recombinant DNA techniques, we inserted three copies of thetetrapeptide (NANP)₃ in the third hypervariable loop (D region)of an immunoglobulin heavy chain variable domain. We show thatthe engineered antibody was properly assembled and secreted.A panel of polyclonal and monoclonal antibodies, including anti-syntheticpeptides and anti-(NANP)_n antibodies, were used to study themolecular configuration of the engineered domain's surface.The results indicate that (i) the exogenous sequence did notappreciably alter the overall fold of the variable domain; and(ii) the inserted epitope folded with a configuration immuno-logicallysimilar to the one assumed in the native protein, suggestingthat short- and medium- rather than long-range interactionsstabilized the structure of the (NANP)₃ peptide in the foldedprotein. We propose this system for the expression of peptidicsequences, and their structural and functional analysis.     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs

The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptide–heavy chain–linker–lightchain–(His)₅ tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 10–30% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with K_d values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay.     

PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome

The A+T-rich genome of the human malaria parasite Plasmodiumfalciparum encodes genes of biological importance that cannotbe expressed efficiently in heterologous eukaryotic systems,owing to an extremely biased codon usage and the presence ofnumerous cryptic polyadenylation sites. In this work we haveoptimized an assembly polymerase chain reaction (PCR) methodfor the fast and extremely accurate synthesis of a 2.1 kb Plasmodiumfalciparum gene (pfsub-1) encoding a subtilisin-like protease.A total of 104 oligonucleotides, designed with the aid of dedicatedcomputer software, were assembled in a single-step PCR. Theassembly was then further amplified by PCR to produce a syntheticgene which has been cloned and successfully expressed in bothPichia pastoris and recombinant baculovirus-infected High Five^TMcells. We believe this strategy to be of special interest asit is simple, accessible and has no limitation with respectto the size of the gene to be synthesized. Used as a systematicapproach for the malarial genome or any other A + T-rich organism,the method allows the rapid synthesis of a nucleotide sequenceoptimized for expression in the system of choice and productionof sufficiently large amounts of biological material for completemolecular and structural characterization.     

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.     

Design and facile production of recombinant resilin-like polypeptides: gene construction and a rapid protein purification method

Resilin is an elastic protein found in specialized regions of the cuticle of insects, which displays unique resilience and fatigue lifetime properties. As is the case with many elastomeric proteins, including elastin, gliadin and spider silks, resilin contains distinct repetitive domains that appear to confer elastic properties to the protein. Recent work within our laboratory has demonstrated that cloning and expression of exon 1 of the Drosophila melanogaster CG15920 gene, encoding a putative resilin-like protein, results in a recombinant protein that can be photochemically crosslinked to form a highly resilient, elastic biomaterial (Rec1 resilin). The current study describes a recursive cloning strategy for generating synthetic genes encoding multiple copies of consensus polypeptides, based on the repetitive domains within resilin-like genes from D. melanogaster and Anopheles gambiae. A simple non-chromatographic purification method that can be applied to these synthetic proteins and Rec1 is also reported. These methods for the design and purification of resilin-like periodic polypeptides will facilitate the future investigation of structural and functional properties of resilin, and the development of novel highly resilient biomaterials.     

Progesterone binding to uteroglobin: two alternative orientations of the ligand

Progesterone binding to a homodimer of uteroglobin takes placein a hydrophobic cavity formed by the two subunits. Previousmutational analyses have shown that the tyrosine (21 and 21')and threonine (60 and 60') residues of the uteroglobin dimerare directly involved in progesterone binding. To analyze thecontribution of each of the two tyrosines and threonines inthe dimer, we have constructed a covalently linked uteroglobindimer (UG^cl) by fusing two uteroglobin cDNAs via a syntheticlinker sequence. Escherichia coli expressed UG^cl bound progesteronewith the same affinity as the native dimeric protein. Replacementof both tyrosines by phenylalanines abolished progesterone binding.Replacement of either the C-terminal tyrosine 21 or the N-terminaltyrosine 21' separately, reduced the affinity for progesterone3- to 4-fold, suggesting that both tyrosines participate inprogesterone binding. In contrast, substitutions of the threonineresidues of the C- or Nterminal moities had no effect, whereasthe replacement of both threonines reduced the affinity forprogesterone 2- to 3-fold. These data, together with computermodels, suggest that progesterone docks in the internal bindingpocket of uteroglobin in two different orientations.     

Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface

Polypeptide library screening technologies are critically dependentupon the characteristics of the expression system employed.A comparative analysis of the lpp–lac, tet and araBAD promoterswas performed to determine the importance of tight regulationand expression level in library screening applications. Thesurface display of single-chain antibody (scFv) in Escherichiacoli as an Lpp–OmpA' fusion was monitored using a fluorescentlytagged antigen in conjunction with flow cytometry. In contrastto the lpp–lac promoter, both tet and araBAD promoterscould be tightly repressed. Tight regulation was found to beessential for preventing rapid depletion of library clones expressingfunctional scFv and thus for maintaining the initial librarydiversity. Induction with subsaturating inducer concentrationsyielded mixed populations of uninduced and fully induced cellsfor both the tet and araBAD expression systems. In contrast,homogeneous expression levels were obtained throughout the populationusing saturating inducer concentrations and could be adjustedby varying the induction time and plasmid copy number. Underoptimal induction conditions for the araBAD system, proteinexpression did not compromise either cell viability or librarydiversity. This expression system was used to screen a libraryof random scFv mutants specific for digoxigenin for clones exhibitingimproved hapten dissociation kinetics. Thus, an expression systemhas been developed which allows library diversity to be preservedand is generally applicable to the screening of E.coli surfacedisplayed libraries.     

Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants

A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesizedand cloned by ligation of 26 deoxyribooligo-nucleotide fragmentsin two steps with a linearized plasmid followed by transformation.On selection by colony hybridization and DNA sequence analysis,clone pTLY.10 was identified to contain a complete T4 lysozymesynthetic DNA. On expression under lac-promoter, unfused T4lysozyme was obtained in {small tilde}4–6% yield. Thedesign and synthesis of two putative folding mutants, flexible(Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75,were based on hierarchical principles. Both mutants lost enzymaticactivity of the wildtype. These results are readily understandableif the hierarchical organization of the structure is taken intoaccount. A possible explanation is that the catalytic sitesare blocked in both mutants.     

The location of inhibitory specificities in human mucus proteinase inhibitor (MPI): separate expression of the COOH-terminal domain yields an active inhibitor of three different proteinases

Human mucus proteinase inhibitor (MPI) consists of 107 aminoacids arranged in two domains showing high homology to eachother. This protein is an inhibitor of different serine proteinasesincluding trypsin, chymotrypsin, leukocyte elastase and cathepsinG. On the basis of sequence comparisons it has been suggestedthat the first domain inhibits trypsin, whereas the second onewas thought to be active against chymotrypsin and elastase.To prove the location of the different inhibitory activitiesgene fragments for both domains have been cloned separatelyand expressed in Escherichia coli. Inhibition assays with theisolated recombinant domains showed that the second domain isactive against chymotrypsin, neutrophil elastase and trypsin,whereas for the first domain only a weak activity against trypsincould be detected. These results suggest that the inhibitoryactivities of the native molecule towards these three proteinasesare all located in the second domain.     

Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps

The gene coding for the tyrosine protein kinase domain of v-fpswas subcloned into a plasmid vector expressing glutathione-S-transferase(GST). This new vector expresses a fusion protein in Escherichiacoli composed of the kinase domain linked with GST at the N-terminus(GST-kin). A portion of the total expressed protein was solubleupon cell lysis and was purified by affinity chromatographyusing glutathione cross-linked agarose. GST-kin (M_r 57 000)is a phosphoprotein as judged by ³²P autoradiography, consistentwith the known autophosphorylation site within the kinase core[Weinmaster et aL (1984) Cell, 37, 559–568]. Cleavageof the fusion protein with thrombin and purification on phosphocelluloseresin yielded the pure kinase domain (M_r 33 000). The activityof the kinase domain is indistinguishable from that of GST-kinusing the peptide substrate EEEIYEEIE, indicating that Nterminalfusion has no effect on the kinase domain. GSTkin phosphorylatesa second peptide, EAEIYEAIE, with improved catalytic efficiency.Initial velocity data are consistent with a random bireactantmechanism with no substrate synergism observed in the ternarycomplex. Steady-state kinetic analyses reveal that this peptideis phosphorylated, with a k_cat of 3.6 s^–1, a K_peptideof 500 µM and a K_ATP of 250 µM. The expression,purification and preliminary kinetic analysis of the kinasedomain of v-fps provide the first step in the application ofstructurefunction studies for this oncoprotein     

Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties

A monomeric variant of triosephosphate isomerase (TIM) witha new engineered binding groove has been characterized further.In this variant (ml8bTIM), the phosphate binding loop had beenshortened, causing the binding site to be much more extended.Here, it is reported that in the V233A variant of ml8bTIM (A-TIM),three important properties of the wild-type TIM active sitehave been restored: (i) the structural properties of loop-7,(ii) the binding site of a conserved water molecule betweenloop-7 and loop-8 and (iii) the binding site of the phosphatemoiety. It is shown that the active site of A-TIM can bind TIMtransition state analogs and suicide inhibitors competently.It is found that the active site geometry of the A-TIM complexesis less compact and more solvent exposed, as in wild-type TIM.This correlates with the observation that the catalytic efficiencyof A-TIM for interconverting the TIM substrates is too low tobe detected. It is also shown that the A-TIM active site canbind compounds which do not bind to wild-type TIM and whichare completely different from the normal TIM substrate, likea citrate molecule. The binding of this citrate molecule isstabilized by hydrogen bonding interactions with the new bindinggroove.     

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.     

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 strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli

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.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

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 proteinA–immunoglobulin interactions.