Bifunctional antibody-Renilla luciferase fusion protein for in vivo optical detection of tumors

An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-positive xenografts, with a tumor:background ratio of 6.0 +/- 0.8 at 6 h after intravenous injection, compared with antigen-negative tumors at 1.0 +/- 0.1 (P = 0.05). Targeting and distribution was also evaluated by microPET imaging using (124)I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.     

Isolation and characterization of mutants of firefly luciferase which produce different colors of light

The luciferase cDNA from the ‘Genji’ firefly, Luciolacruciata, was mutated with hydroxylamine to isolate mutant lucierases.Some of the isolated mutant enzymes produced different colorsof light, ranging from green to red. Five such mutants, producinggreen (_max = 558 nm), yellow-orange (_max = 595 nm), orange (_max= 607 nm) and red light (_max = 609 and 612 nm), were analyzed.The mutations were found to be single amino acid changes, fromVal239 to IIe, Pro452 to Ser, Ser286 to Asn, Gly326 to Ser andHis433 to Tyr respectively.     

In vivo system for the detection of low level activity barnase mutants

In previous studies, the replacement of His35 in the pore-formingprotein -hemolysin (HL) with Leu, lie, Pro, Arg, Ser, Thr orCys yielded inactive polypeptides. Here, we show that modificationof the inactive single-cysteine mutant HL-H35C with iodoacetamide,to form H35CamC, generates significant pore-forming activity.The closely related polypeptides H35N and H35Q have, respectively,essentially no activity and greatly reduced activity. The modifiedresidue in H35CamC, S-carboxamidomethylcysteine, mimicks histidinein volume, polarity and hydrogen bonding potential, but is unableto ionize. Unmodified H35C is defective in the final step ofpore formation: the conversion of an inactive heptameric membrane-boundassembly intermediate to a structure containing open channels.It is this step in assembly, that is ameliorated in H35CamC.     

Humanization of a mouse anti-human IgE antibody: a potential therapeutic for IgE-mediated allergies

Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and,therefore, has potential as a therapeutic agent in patientswith IgE-mediated allergies such as hay fever, food and drugallergies and extrinsic asthma. The clinical usefulness of mouseantibodies is limited, however, due to their immunogenidty inhumans. Mouse C21 antibody was humanized by complementaritydetermining region (CDR) grafting with the aim of developingan effective and safe therapeutic for the treatment of IgE-mediatedallergies. The CDR-grafted, or reshaped human, C21 variableregions were carefully designed using a specially constructedmolecular model of the mouse C21 variable regions. A key stepin the design of reshaped human variable regions is the selectionof the human framework regions (FRs) to serve as the backbonesof the reshaped human variable regions. Two approaches to theselection of human FRs were tested: (i) selection from humanconsensus sequences and (ii) selection from individual humanantibodies. The reshaped human and mouse C21 antibodies weretested and compared using a biosensor to measure the kineticsof binding to human IgE. Surprisingly, a few of the reshapedhuman C21 antibodies exhibited patterns of binding and affinitiesthat were essentially identical to those of mouse C21 antibody.     

A rapid and effective procedure for screening protease mutants

We describe a simple and effective procedure to screen for activeproteases among a large number of mutants. First, the mutantsare genetically tested by the protease activity produced inthe periplasm of transformed bacteria which supplies the cellswith a nitrogen source by hydrolyzing a protein applied to plates.Then a less sensitive activity staining and an X-ray film digestionassay are used to verify and estimate the activity of the mutantsthat proved to be positive in the first step. Depending essentiallyon the level of periplasmic protease activity, the method candetect both the activity and the stability of the expressedenzymes. We calibrated the method with transformants that producewild-type trypsin, chymotrypsin and trypsin mutants of knownactivity. Using this method we found two active revertants ofthe inactive Asn102 trypsin mutant, by screening {small tilde}4.4x10⁴random mutants that were generated by the polymerase chain reactionon a cDNA fragment. This procedure should be useful in searchingfor proteases of novel specificity and/or reaction chemistryengineered by random mutagenesis, and also for in vitro evolutionstudies.     

Digital screening methodology for the directed evolution of transglycosidases

Engineering of glycosidases with efficient transglycosidasesactivity is an alternative to glycosyltransferases or glycosynthasesfor the synthesis of oligosaccharides and glycoconjugates. However,the engineering of transglycosidases by directed evolution methodologiesis hampered by the lack of efficient screening systems for sugar-transferactivity. We report here the development of digital imaging-basedhigh-throughput screening methodology for the directed evolutionof glycosidases into transgalactosidases. Using this methodology,we detected transglycosidase mutants in intact Escherichia colicells by digital imaging monitoring of the activation of non-or low-hydrolytic mutants by an acceptor substrate. We screenedseveral libraries of mutants of β-glycosidase from Thermusthermophilus using this methodology and found variants withup to a 70-fold overall increase in the transglycosidase/hydrolysisactivity ratio. Using natural disaccharide acceptors, thesetransglycosidase mutants were able to synthesise trisaccharides,as a mixture of two regioisomers, with up to 76% yield.     

Comparative stability of dihydrofolate reductase mutants in vitro and in vivo

Dihydrofolate reductase mutants with amino acid replacementsin the active center (Thr35 Asp mutant, Arg57 His mutant andthe mutant with triple replacement Thr35 Asp, Asn37 Ser, Arg57 His) were obtained by site-directed mutagenesis. The stabilizationeffect of trimethoprim and NADP·H on the protein tertiarystructure in vitro has been investigated. In the case of mutantswith a ‘weak’ tertiary structure (Thr35 Asp35 andthe triple mutant) the separate addition of ligands does notaffect their stability. The simultaneous addition of these ligandsto Thr35 Asp35 and the triple mutant leads to the large increasein their stability. A distinct correlation was found betweenthe in vitro studied stability of the mutant proteins to theurea- or heat-induced denaturation and the level of proteolyticdegradation of these mutants previously observed in vivo.     

A single amino acid substitution can restore the solubility of aggregated colicin A mutants in Escherichia coli

Mutants of colicin A have been prepared in which the three tryptophanresidues (Trp86, Trpl30 and Trpl40), localized in the C-terminaldomain of the soluble wild-type protein, have been substitutedby phenylalanine. The Trpl40Phe single mutation had the effectof decreasing the percentage of protein that is expressed asinsoluble aggregates. The created hydrophobic cavity decreasedthe stability of the protein during its folding, resulting inpartial aggregation in the cytoplasm of the Escherichia coli-producingcells. Aggregation was increased when Trpl40 was substitutedby Lys, Leu or Cys, or if the Trpl40 mutation was combined withthe Trp86Phe and/ or Trpl30Phe mutations. A single mutation,Lysll3Phe, however, was able to restore the solubility of theaggregated mutants in vivo. Detailed analysis of the 3-D structureof the C-terminal domain of colicin A suggests that fillingof the hydrophobic cavity is responsible for this effect.     

Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis

Docking models of fructosyl amine oxidase (FAOD) from the marineyeast Pichia N1-1 (N1-1 FAOD) with the substrates fructosylvaline (f-Val) and fructosyl-N-lysine (f-Lys) were producedusing three-dimensional protein model as reported previously(Miura et al., 2006, Biotechnol. Lett., 28, 1895-1900). Theresidues involved in recognition of substrates were proposed,particularly Asn354, which interacts closely with f-Lys, butnot with f-Val. Substitution of Asn354 to histidine and lysinesimultaneously resulted in an increase in activity of f-valand a decrease in activity of f-Lys and thus, increasing thespecificity for f-Val from 13- to 19-fold. In addition to creatingtwo mutant FAODs with great potential for the measurement ofglycated hemoglobin, we have provided the first structural modelof substrate binding with eukaryotic FAOD, which is expectedto contribute to further investigation of FAOD.     

A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility

Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.     

Generation and analysis of proline mutants in protein G

The pyrrolidine ring of the amino acid proline reduces the conformational freedom of the protein backbone in its unfolded form and thus enhances protein stability. The strategy of inserting proline into regions of the protein where it does not perturb the structure has been utilized to stabilize many different proteins including enzymes. However, most of these efforts have been based on trial and error, rather than rational design. Here, we try to understand proline's effect on protein stability by introducing proline mutations into various regions of the B1 domain of Streptococcal protein G. We also applied the Optimization of Rotamers By Iterative Techniques computational protein design program, using two different solvation models, to determine the extent to which it could predict the stabilizing and destabilizing effects of prolines. Use of a surface area dependent solvation model resulted in a modest correlation between the experimental free energy of folding and computed energies; on the other hand, use of a Gaussian solvent exclusion model led to significant positive correlation. Including a backbone conformational entropy term to the computational energies increases the statistical significance of the correlation between the experimental stabilities and both solvation models.     

X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A

The structures of the single-residue mutants H134Q and Y76Aof bovine pancreatic DNase I have been determined and refinedincluding data to 2.3 and 2.4 Å resolution respectively,by X-ray crystallography. H134 is an essential catalytic residue,while Y76 contributes to the binding of DNA by providing a largevan der Waals contact area that stabilizes the wide minor grooveseen in DNase I-DNA complexes. The mutant proteins, which showstrongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A),were expressed in E.coli and both crystallize in space-groupC2 with almost identical unit cells. The crystal packing schemeis different from that found in wild type crystals grown undervery similar conditions, presumably due to the absence of thecarbohydrate moiety. In both mutants the conformation of theprotein is nearly identical to that of the wild type enzymeand changes are confined to surface loops involved in packing.The disruption of the hydrogen bonds between H134, E78 and Y76in both mutants leads to an increased mobility and positionalshifts in the DNA-binding loop, mainly around residue Y76. Thisin turn may further reduce DNA-binding affinity and, thus, contributeto the low activity. In contrast, symmetry contacts involvingresidues 97–108 lead to a stabilization of the flexibleloop compared to wild type DNase I.     

Protein stability for single substitution mutants and the extent of local compactness in the denatured state

The stability changes caused by single amino acid substitutionsare studied by a simple, empirical method which takes accountof the free energy change in the compact denatured state aswell as in the native state. The conformational free energyis estimated from effective inter-residue contact energies,as evaluated in our previous study. When this method is applied,with a simple assumption about the compactness of the denaturedstate, for single amino acid replacements at Glu49 of the tryptophansynthase subunit and at Ile3 of bacteriophage T4 lysozyme,the estimates of the unfolding Gibbs free energy changes correlatewell with observed values, especially for hydrophobic aminoacids, and it also yields the same magnitudes of energy as theobserved values for both proteins. When it is also applied foramino acid replacements at various positions to estimate theaverage number of contacts at each position in the denaturedstate from the observed value of unfolding free energy change,those values for replacements with Gly and Ala at the same residueposition in staphylococcal nuclease correlate well with eachother. The estimated numbers of contacts indicate that the proteinis not fully expanded in the denatured state and also that thecompact denatured state may have a substantially native-liketopology, like the molten globule state, in that there is aweak correlation between the estimated average number of contactsat each residue position in the denatured state and the numberof contacts in the native structure. These results provide somefurther evidence that the inter-residue contact energies asapplied here (i) properly reflect actual inter-residue interactionsand (ii) can be considered to be a pairwise hydrophobicity scale.Also, the results indicate that characterization of the denaturedstate is critical to understanding the folding process.     

Structure prediction of subtilisin BPN' mutants using molecular dynamics methods

In this paper we describe the achievements and pitfalls encounteredin doing structure predictions of protein mutants using moleculardynamics simulation techniques in which properties of atomsare slowly changed as a function of time. Basically the methodconsists of a thermodynamic integration (slow growth) calculationused for free energy determination, but aimed at structure prediction;this allows for a fast determination of the mutant structure.We compared the calculated structure of the mutants Met222Ala,Met222Phe and Met222Gln of subtilisin BPN' with the respectiveX-ray structures and found good agreement between predictedand X-ray structure. The conformation of the residue subjectto the mutation is relatively easy to predict and is mainlydetermined by packing criteria. When the side chain has polargroups its exact orientation may pose problems; long-range Coulombinteractions may generate a polarization feedback involvingsystem relaxation times beyond the simulation time. Changesinduced in the environment are harder to predict using thismethod. In particular, rearrangement of the hydration structurewas difficult to predict correctly, probably because of thelong relaxation times. In all conversions made the changes observedin the environment were found to be history-dependent and inparticular the hydrogen bonding patterns provided evidence formetastable substates. In all cases the structure predicted wascompared with available kinetic data and the reduced activitycould be explained in terms of changes in the configurationof the active site.     

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11

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.     

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.     

Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme

Five different cysteine-containing mutants of the lysozyme frombacteriopbage T4 were used to explore the feasibility of usingsite-directed mutagenesis to generate isomorphous heavy-atomderivatives for protein crystallography. Cysteines 54 and 97,present in wild-type lysozyme, can be readily reacted with mercuricion to produce an excellent isomorphous heavy-atom derivative.Mutants with an additional cysteine at position 86,146,153 or157, or with Cys 97 replaced by Val, were engineered by site-directedmutagenesis. The mutant lysozyme Thr 157 - Cys reacts with mercuricchloride to give an excellent new derivatve although Cys 157is only -60% substituted with the heavy atom. The cysteine atposition 146 is largely buried but reacts readily with mercuricchloride. In this case the isomorphism is poor and the resultantderivative is of marginal quality. Cys 153 reacts rapidly withmercuric ion but the derivative crystals do not diffract. Themutant Pro 86 - Cys does not yield a particularly good heavy-atomderivative. This is due in part to a loss of isomorphism associatedwith the mutation. In addition, Cys 86 shows very little reactivitytowards mercurials even though it is fully exposed to solvent.The mutation Cys 97 Val was used to explore the possibilityof creating an independent derivative by deleting a heavy-atomsite already present in wild-type lysozyme. In all cases thatwere tested, the quality of the heavy-atom derivative was improvedby using as an isomorphous pair mercury-substituted mutant versusnon-substituted mutant rather than mercury-substituted mutantversus (non-substituted) wild-type lysozyme. Unexpectedly, thecysteines that are most exposed to solvent and most mobile areleast reactive toward mercuric chloride. The cysteines thatprovide the best heavy-atom sites are those that are locatedin surface crevices and are only partly exposed to solvent.     

A dual-affinity gene fusion system to express small recombinant proteins in a soluble form: expression and characterization of protein A deletion mutants

A novel gene fusion system to express and purify small recombinantproteins in Escherichia coli has been constructed. The conceptallows for affinity purification of soluble gene products bysequential albumin- and Zn²+-affinity chromatography. The dual-affinitysystem is well suited for expression of unstable proteins asonly full-length protein is obtained after purification andproteins gain proteolytic stability in the fusion protein. Herewe show that the dual-affinity approach can be used for theexpression of various unstable derivatives of a single IgG-bindingdomain based on staphylococcal protein A. Analysis of the proteolyticstabilities and the IgG-binding properties of the differentmutant proteins suggest that the model for the structure ofan IgG-binding domain must be re-evaluated.     

Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase

The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.     

Expression of functional papain precursor in Saccharomyces cerevisiae: rapid screening of mutants

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.