Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (V(H) and V(L)) domains of two different specificities connected by three linkers. When assembled in the order V(H)(A)-linker(1)-V(L)(B)-linker(2)-V(H)(B)-linker(3)-V(L)(A), the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary V(H) and V(L) domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains. We generated a number of single-chain constructs using four V(H) and V(L) domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6-27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites.     

Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six differentlinker peptides to join the V_H and V_L domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)₃ linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcore^TM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric.     

Rational design of a chimeric endonuclease targeted to NotI recognition site

A cleavage-deficient variant of NotI restriction endonuclease (GCGGCCGC) was isolated by random mutagenesis of the notIR gene. The NotI variant D160N was shown to bind DNA and protect plasmid DNA from EagI (CGGCCG) and NotI digestions. The EDTA-resistant BmrI restriction endonuclease cleaves DNA sequence ACTGGG N5/N4. The N-terminal cleavage domain of BmrI (residues 1-198) with non-specific nuclease activity was fused to the NotI variant D160N with a short linker. The engineered chimeric endonuclease (CH-endonuclease) recognizes NotI sites specifically in the presence of high salt (100-150 mM NaCl) and divalent cations Mg++ or Ca++. In contrast to wild-type NotI, which cuts within its recognition sequence, BmrI198-NotI (D160N) cleaves DNA outside of NotI sites, resulting in deletion of the NotI site and the adjacent sequences. The fusion of the BmrI cleavage domain to cleavage-deficient variants of Type II restriction enzymes to generate novel cleavage sites will provide useful tools for DNA manipulation.     

Expression and enzymatic characterization of the soluble recombinant plasmepsin I from Plasmodium falciparum

The plasmepsins are involved in the degradation of host cell hemoglobin during malaria infection. Plasmepsin I (PM I) initiates the degradative process, and has been suggested as an attractive target for the treatment of malaria. The production of active recombinant PM I, however, has been challenging. We report for the first time, the expression and partial characterization of soluble recombinant PM I from Plasmodium falciparum in which a truncated form of PM I (Lys77P-Leu329) (P indicates a propart residues) was fused to thioredoxin in the pET32b(+) vector, Trx-tPM I and expressed in Escherichia coli Rosetta-gami B (DE3)pLysS. The soluble fusion protein was purified from cell culture using a combination of Ni(2+) affinity and gel filtration chromatography and was capable of autocatalytic activation at pH 4.0-5.5, which occurred at Leu116P-Ser117P, seven residues upstream of the native cleavage site (Gly123P-Asn1). The mature tPM I (mtPM I) was capable of hydrolyzing both human hemoglobin with a pH optimum of pH 2.8-4.0 and the synthetic fluorogenic peptide EDANS-CO-CH(2)-CH(2)-CO-ALERMFLSFP-Dap(DABCYL)-OH with a dual pH optima of pH 2.5-3.0 and pH 4.5-5.5. Using the synthetic substrate, mtPM I exhibited kinetic parameters comparable to native PM I.     

Protein modelling using a chimera reference protein derived from exons

Bovine pancreatic /S-trypsin (PDB ID-code: 1TPO) which is registeredin the Brookhaven Protein Data Bank (PDB) consists of four exons.The results of homology searches for each exon in the PDB showedthat homologous proteins were tonin (PDB ID-code: 1TON), ratmast cell protease (PDB ID-code: 3RP2_A), kaffikrein A (PDBID-code: 2PKA_B) and kallikrein A (2PKA_B) respectively. Thus,for the three-dimensional structure prediction of 1TPO, a chimeraprotein was constructed from the three proteins mentioned aboveand the 3-D structure prediction was performed using this chimerareference protein. The modelled structure of 1TPO was energeticallyoptimized by molecular mechanics and molecular dynamics simulationand was compared with its X-ray crystal structure registeredin the PDB. The root mean square deviations (r.m.s.d.) of mainchain atoms and the neighbouring active site (5 sphere fromHis57, AsplO2 and Serl95) between the modelled structure andthe X-ray structure were 1.66 and 0.94 respectively. Porcinepancreatic elastase (PDB ID-code: 3EST) which is registeredin the PDB was used as the reference protein and the modelledstructure from 3EST was also compared with the X-ray data. Ther.m.s.d. of main chain atoms and that of the active site were2.14 and 1.18 respectively. These results dearly support thepropriety of this method using the chimera reference protein.     

Variants of human tissue-type plasminogen activator substituted at the protease cleavage site and glycosylation sites, and truncated at the N- and C-termini

Mutations were directed to specific regions of the human tissue-typeplasminogen activator (t-PA) gene in an effort to better definestructure–function relationships of the enzyme. Threetypes of modifications were effected by in vitro mutagenesis:elimination of glycosylation sites; substitutions of amino acidsat the cleavage site for conversion of single-chain t-PA totwo-chain t-PA; and truncations of the N- and C-termini. Thirteenvariants were purified from permanent CHO cell lines and analyzedfor specific activity, fibrin stimulation, fibrin binding, inhibitionby plasminogen activator inhibitor-2 (PAI-2) and half-life.The results of these analyses are: (i) variants with carbohydrate–depletedkringle domains possessed higher specific activities than wild-typet-PA; (ii) a cleavage site variant substituted at Arg275 withGly had greatly reduced specific activity; (iii) two variantssubstituted at Lys277 exhibited altered interactions with PAI-2;(iv) the variant with a truncated C-terminus had reduced activityin the absence of fibrin; and (v) no variants had significantlyaltered half–lives. In order to test the effects of combiningmutations, four additional variants were produced. Each combinationvariant retained at least one of the altered properties observedin the original variants, and in three of the variants the diverseproperties were additive.     

Prediction of signal peptides in archaea

Computational prediction of signal peptides (SPs) and theircleavage sites is of great importance in computational biology;however, currently there is no available method capable of predictingreliably the SPs of archaea, due to the limited amount of experimentallyverified proteins with SPs. We performed an extensive literaturesearch in order to identify archaeal proteins having experimentallyverified SP and managed to find 69 such proteins, the largestnumber ever reported. A detailed analysis of these sequencesrevealed some unique features of the SPs of archaea, such asthe unique amino acid composition of the hydrophobic regionwith a higher than expected occurrence of isoleucine, and acleavage site resembling more the sequences of gram-positiveswith almost equal amounts of alanine and valine at the position-3before the cleavage site and a dominant alanine at position-1,followed in abundance by serine and glycine. Using these proteinsas a training set, we trained a hidden Markov model method thatpredicts the presence of the SPs and their cleavage sites andalso discriminates such proteins from cytoplasmic and transmembraneones. The method performs satisfactorily, yielding a 35-foldcross-validation procedure, a sensitivity of 100% and specificity98.41% with the Matthews’ correlation coefficient beingequal to 0.964. This particular method is currently the onlyavailable method for the prediction of secretory SPs in archaea,and performs consistently and significantly better comparedwith other available predictors that were trained on sequencesof eukaryotic or bacterial origin. Searching 48 completely sequencedarchaeal genomes we identified 9437 putative SPs. The method,PRED-SIGNAL, and the results are freely available for academicusers at http://bioinformatics.biol.uoa.gr/PRED-SIGNAL/ andwe anticipate that it will be a valuable tool for the computationalanalysis of archaeal genomes.     

Mammalian cell transient expression of tissue factor for the production of antigen

We describe a mammalian cell expression system used to rapidlyproduce microgram quantities of a membrane protein used as animraunogen. A fusion protein expression vector was constructedwhich contained the signal sequence and 27 amino acids of theHerpes simplex virus glycoprotein D (gD), followed by a factorVIII (fVIII) thrombin cleavage site and the mature tissue factor(TF) sequence. This fusion protein was transiently expressedand then purified using an antibody to gD. The purified fusionprotein, gDTF, was incubated with thrombin to remove the gD-fVIIImoiety and the resulting rTF served as antigen for the generationof TF-specific antibodies. The antibodies produced were thenused for a comparison of the turnover rates of the constitutivelyand transiently produced fusion protein. In addition, sensitivityto glycosidases indicated that the transiently and constitutivelyproduced recombinant proteins do not contain identical carbohydratestructures.     

Multimerization behaviour of single chain Fv variants for the tumour-binding antibody B72.3

A systematic study has been performed on the relationship betweenlinker length, relative orientation of variable domains, multimerizationbehaviour and antigen binding activity for single chain Fvs(scFvs) of the tumour-binding antibody B72.3. Thirteen scFvvariants with linkers comprising up to six repeats of the motifGly-Gly-Gry-Gly-Ser were studied. All these scFvs showed a tendencyto form dimers or higher molecular weight species, and thistendency decreased with increasing linker length. The dimersand higher molecular weight forms may arise from head to tailintermolecular association of V_H and V_L domains. For each linkerlength, scFvs with the organization VL-linker-VH showed greaterbinding activity than those with the organization VH-linker-VL.In fact, for the latter organization only the variant with a30 amino acid linker showed good binding activity, suggestingthat (0 for B72.3 the C-tenninus of VH or the N-tenninus ofVL makes a structural contribution to antigen binding, and (ii)shorter linkers interfere with this contribution. Antigen bindingstudies on scFvs should be interpreted with caution becauseof their tendency to multlmerize. Such multimerization can beminimized by using linkers longer than those in common use     

A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli

Thermolysin, a representative zinc metalloproteinase from Bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. The conventional expression method for recombinant thermolysin requires the autocatalytic cleavage, so that production of a mutant thermolysin is affected by its autocatalytic digestion activity. In this study, we have established a new expression method that does not require the autocatalytic cleavage. The mature sequence of thermolysin containing an NH(2)-terminal pelB leader sequence and the pre-prosequence of thermolysin were co-expressed constitutively in Escherichia coli as independent polypeptides under the original promoter sequences in the npr gene which encodes thermolysin. Unlike the conventional expression method, not only the wild-type thermolysin but also mutant thermolysins [E143A (Glu143 is replaced with Ala), N112A, N112D, N112E, N112H, N112K and N112R] were produced into the culture medium. The wild-type enzyme expressed in the present method was indistinguishable from that expressed in the conventional method based on autocatalytic cleavage, as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester. The present method should be useful especially for preparation of active-site mutants of thermolysin, which might have suppressed autocatalytic digestion activity. The results also demonstrate clearly that the covalent linking between the pro- and mature sequences is not necessary for the proper folding of the mature sequence by the propeptide in thermolysin.     

Stabilized variant of Streptomyces subtilisin inhibitor and its use in stabilizing subtilisin BPN'

Protein protease inhibitors could potentially be used to stabilize proteases in commercial products such as liquid laundry detergents. However, many protein protease inhibitors are susceptible to hydrolysis inflicted by the protease. We have engineered Streptomyces subtilisin inhibitor (SSI) to resist proteolysis by adding an interchain disulfide bond and removing a subtilisin cleavage site at leucine 63. When these stabilizing changes were combined with changes to optimize the affinity for subtilisin, the resulting inhibitor provided complete protease stability for at least 5 months at 31 degrees C in a subtilisin-containing liquid laundry detergent and allowed full recovery of the subtilisin activity upon the dilution that occurs in a North American washing machine.     

Efficient site specific removal of a C-terminal FLAG fusion from a Fab' using copper(II) ion catalysed protein cleavage

The peptide sequence ^NDKTH^C was investigated as a site for efficient,specific cleavage of a fusion protein by cupric ions using ahumanised 1 Fab' as a model protein. The native upper hinge^NDKTH^C sequence was mutated to create a site resistant to cleavageby cupric ions and a ^NDKTH^C sequence introduced between thehinge and a C-terminal FLAG peptide. Incubation of Fab' withCu²⁺ at 62°C at alkaline pHs resulted in removal of theFLAG peptide with efficiencies of up to 86%. Cleavage conditionsdid not detrimentally affect the Fab' protein. Use of the ^NDKTH^Csequence along with cupric ions may provide a cost-effectivemethod for large scale proteolytic cleavage of fusion proteins.     

Inhibitory mode of N-phenyl-4-pyrazolo[1,5-b] pyridazin-3-ylpyrimidin-2-amine series derivatives against GSK-3: molecular docking and 3D-QSAR analyses

Glycogen synthase kinase 3 (GSK-3) inhibition is an important research topic because of its wide range of associated health implications. The interaction mode of a series of N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amine compounds with human GSK-3 has been studied using molecular docking and 3D-QSAR approaches. In the 3D-QSAR studies, the molecular alignment and conformation determination are so important that they affect the success of a model. Flexible docking (AutoDock3.0.5) was used for the determination of 'active' conformation and molecular alignment. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used to develop 3D-QSAR models of 80 N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amine compounds. The r(2) values were 0.870 and 0.861 for CoMFA and CoMSIA models, respectively. The predictive ability of these models was validated by 10 compounds of the test set. Mapping these models back to the topology of the active site of GSK-3 led to a better understanding of the vital N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amines-GSK-3 interactions. The results demonstrate that combination of ligand-based and receptor-based modeling is a powerful approach to build 3D-QSAR models. The interaction mode from this study may be helpful for the design of a novel inhibitor and guide the selection of candidate sites for further experimental studies on site-directed mutagenesis.     

Rational design and selection of bivalent peptide ligands of thrombin incorporating P4-P1 tetrapeptide sequences: from good substrates to potent inhibitors

The tetrapeptide Phe-Asn-Pro-Arg is a structurally optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or some variants to a C-terminal fragment of hirudin, we were able to generate a series of new bivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P3 and P3' sites of the active-site directed sequence, Phe(P4)-Xaa(P3)-Pro(P2)-Arg(P1)-Pro(P1')-Gln(P2')-Yaa(P3'). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3' sites for bivalent and optimized two-site binding. Very significantly, panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-clotting activities in the low nanomolar range and yet interfering only partially the catalytic active site of thrombin. Modes of action of the newly discovered bivalent inhibitors are rationalized in light of the allosteric properties of thrombin, especially the interplay between the proteolytic action and regulatory binding occurring at thrombin surfaces remote from the catalytic active site.     

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     

Pseudomonas glumae lipase: increased proteolytic stabifity by protein engineering

The feasibility of stabilizing proteins towards proteolyticdegradation was explored by engineering the primary proteolyticcleavage site(s). This novel approach does not require informationon the 3-D structure of the native enzyme. As a model system,the extracellular lipase of Pseudomonas glumae was chosen, whichis sensitive towards degradation by subtilisin-tvpe proteases.The primary proteolytic cleavage in the lipase appeared to belocated between amino acids serine 153 and histidine 154. SincesubtUisins are known to show a preference towards amino acidresidues surrounding the scissile bond, non-preferred aminoadds were introduced in this area. Two concepts were tested:the introduction of arginine or glutainate residues (chargeconcept) and the introduction of proline residues (proUne concept).Although the mutant Upases produced according to either of theseconcepts were still cleaved in the same area, they showed aconsiderably increased stability towards proteolytic degradation.     

Energy calculations and analysis of HIV-1 protease-inhibitor crystal structures

The interactions between HIV–1 protease and its boundinhibitors have been investigated by molecular mechanics calculationsand by analysis of crystal structures of the complexes in orderto determine general rules for inhibitor and substrate bindingto the protease. Fifteen crystal structures of HTV–1 proteasewith different peptidomimetic inhibitors showed conservationof hydrogen bond interactions between the main chain C=O andNH groups of the inhibitors and the C=O and NH groups of theprotease extending from P3 C=O to P3' NH. The mean length ofthe hydrogen bonds between the inhibitor and the flexible flapsand the conserved water molecule (2.9 À) is slightlyshorter than the mean length of hydrogen bonds between the inhibitorand the more rigid active site region (3.1 À) of theprotease. The two hydrogen bonds between the conserved waterand P2 and P1' carbonyl oxygen atoms of the inhibitor are theshortest and are predicted to be important for the tight bindingof inhibitors. Molecular mechanics analysis of three crystalstructures of HIV-1 protease with different inhibitors withindependent calculations using the programs Discover and Brugelgave an estimate of 56-68% for the contribution of all the inhibitormain chain atoms to the total calculated protease–inhibitorinteraction energy. The contribution of individual inhibitorresidues to the interaction energy wascalculated using Brugel.The main chain atoms of residue P2 had a consistently largefavorable contribution to the total interaction energy, probablydue to the presence of the two short hydrogen bonds to the flexibleflap. The contribution of individual inhibitor side chains dependedon the size of the side chain and the presence of specific hydrogenbond interactions with the protease.     

Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli

The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamideacetyltransferase subunits (E2p) of the Escherichia coli pyruvatedehydrogenase complex are connected by linker sequences whichare characteristically rich in alanine and proline residues.By facilitating domain movement these linkers are thought topromote interactions between the three types of active sitethat participate in the catalytic cycle of the complex. To investigatefunctional constraints associated with linker composition andsequence, the natural linker of an E2p subunit containing onelipoyl domain was replaced by shorter sequences containing:mixtures of alanine plus proline residues; mainly alanine; mainlyproline; and mainly charged residues. Each artificial linkerpossessed a central histidine residue for assessing linker flexibilityby ¹H-NMR spectroscopy. The resultant complexes exhibited 181%(proline), 74–79% (alanine plus proline), 63% (alanine)and 7% (charged residues) of parental activity compared witha value of 75% expected for a complex with a comparably shortenedlinker. The ¹H-NMR spectra showed that the alanine plus prolinelinkers are flexible but the alanine linker and the prolinelinker are relatively inflexible. Substantial variations inlinker sequence and composition were tolerated without lossof function, and the enhanced activity conferred by the prolinelinker was attributed to the combined effects of length andrelative inflexibility.     

Expression in insect cells and purification of a catalytically active recombinant human gastric lipase

Human gastric lipase (HGL) cDNA was synthesized by RT-PCR amplificationand cloned into the PVL 1392 baculovirus transfer vector. Therecombinant transfer vector was cotransfected with a modifiedbaculovirus DNA (Baculogold^TM) which contains a lethal deletion.Cotransfection of baculovirus DNA with the recombinant transfervector rescues the lethal deletion of this virus DNA and reconstitutesviable virus particles inside the transfected insect cells.BTI-TN-5B1-4 insect cells (also called High Five^TM cells) wereused to express recombinant HGL. The level of HGL secretionwas {small tilde}32 mg/1 of culture medium. The insect cellsalso accumulated HGL intracellularly, which indicated the existenceof rate-limiting steps in the secretion of HGL. Therefore weinvestigated the effect of replacing the HGL signal peptide(SP) by other SP of secreted proteins. The honeybee melittinSP and the human pancreatic lipase (HPL) SP were tested. Thefusion of HGL with HPL SP resulted in a 2-fold increase in theamount of lipase secreted from the insect cells. The recombinantactive HGL was not processed at the expected cleavage site ofthe natural enzyme, however, but at residue +3. On the otherhand, High Five^TM cells transfected with the vector encodingHGL fused to the melittin SP did not secrete any detectableactive HGL. Recombinant HGL was identified using the Westernblot procedure with rabbit polyclonal antibodies. The proteinmigrated with an apparent molecular mass of 45 kDa under SDS–PAGEanalysis (compared with 50 kDa in the case of natural HGL),indicating that the insect cells have only a limited capacityto glycosylate HGL. The maximum specific activities of the recombinantlipase were 434, 730 and 562 units/mg using long-chain (Intralipid^TM),medium-chain (trioctanoylglycerol) and short-chain (tributyroylglycerol)triacylglycerols, respectively.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84are each replaced by a Cys residue, it should be possible toform a third disulfide bond in ribonuclease T1 (RNase T1) betweenthese residues, with only minimal conformational changes atthe catalytic site. The gene encoding such a mutant variantof RNase T1 (Tyr24 – Cys24, Asn84 – Cys84) was constructedby the cassette mutagenesis method using a chemically synthesizedgene. In order to reduce the toxic effect of the mutant enzyme(RNase T1S) on an Escherichia coli host, we arranged for theprotein to be secreted into the periplasmic space by using avector that harbors a gene for an alkaline phosphatase signalpeptide under the control of the trp promoter. The nucleolyticactivity of RNase T1S toward pGpC was approximately the sameas that of RNase T1 at 37°C (pH 7.5). Moreover, at 55°C,RNase T1S retained nearly 70% of its activity while the activityof the wild-type enzyme was reduced to <10%. RNase T1S wasalso more resistant to denaturation by urea than the wild-typeenzyme. However, unlike RNase T1, RNase T1S was irreversiblyand almost totally inactivated by boiling at 100°C for 15min.