Prion protein gene expression in cultured cells

A single copy gene encodes both the scrapie (PrP^Sc) and cellular(PrP^C) isoforms of the prion protein (PrP). Cultured cell lineswere found to express the endogenous PrP mRNA at levels comparableto those observed in the brains of adult rodents; however, thesecells were invariably found to express greatly reduced levelsof PrP. In all the cell lines examined, PrP was undetectableby Western immunoblot analysis. These cells were also poor recipientsfor expression constructs linking the hamster PrP gene openreading frame to several strong eukaryotic promoters; stableclones derived by transfection of these expression vectors failedto show elevated expression of PrP. When extremely high levelsof PrP mRNA were produced using either an insect baculovirusor a mammalian SV40 based vector, significant quantities ofPrP were produced, although in both cases the proteins wereapparently processed differently from the PrP^C observed in brains.In an expression system using an SV40 late promoter vector inmonkey COS-7 cells, a significant fraction of PrP was transportedto the cell surface where PrP^C is found in vivo. PrP synthesizedby the baculovirus vector failed to induce scrapie in hamstersand did not possess the characteristics of the PrP^Sc isoformassociated with infectivity. The SV40 late promoter vector systemmay permit experiments designed to elucidate the role of PrP^Scduring scrapie infection as well as the function of PrP^C innormal metabolism.     

Expression of synthetic genes coding for completely new, nutritionally rich, artificial proteins

Synthetic genes (A, AB and AHB) constructed and cloned intopKK233-2 vector were recloned from the parent plasmid into thenew procaryotic expression vectors pGFY221N and pBIO52. GeneAF^–B (coding for all amino acids besides phenylalanine)was obtained by ‘cassette mutagenesis’ from geneAB. The plasmid pGFY221N was constructed from pGFY218L by replacingthe PstI by an NcoI site; plasmid pBIO52 was derived from pGFY221Nthrough replacing the 221-bp EoRl/NcoI fragment with a syntheticDNA segment of 52 bp representing the Escherichia coli atpEgene translational initiation region. The genes A, AB, AHB andAF^–B in the vector pGFY221N were expressed with a six-amino-acid-longleader sequence; in pBIO52 the genes were expressed directly.in vitro expression experiments were successful with all thegenes except with the AHB gene integrated into pGFY221N. Inthe E. coli minicell system expression was demonstrated withthe A gene in pGFY221N and the AF^–B and AHB genes in pBIO52.Complete translation of the expressed genes AB, AF^–B andAHB in either the in vitro or in vivo systems could be shownby using ³⁵S-labelled N-terminal methionine and C-terminal cysteine.Both amino acids occur only once in the peptide sequences.     

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

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

Domain-domain interactions in hybrids of tissue-type plasminogen activator and urokinase-type plasminogen activator

Fibrin-dependent plasminogen activation by tissue-type plasminogenactivator (t-PA) is in part associated with the presence ofthe kringle 2 domain in t-PA. Within this kringle 2 domain alysyl-binding site has been described. The plasminogen to plasminconversion by urokinase-type plasminogen activator (u-PA), incontrast to that of t-PA, is not enhanced in the presence offibrin. Within the u-PA kringle domain no lysyl-binding siteis found. To study whether introduction of a lysyl-binding sitein the u-PA kringle domain will make u-PA a fibrin-dependentplasminogen activator, three stretches of amino acid residuesof the u-PA kringle domain (A²⁸-Q³³, D⁵⁵-N⁵⁷ and G⁶⁷-V⁷²) weresubstituted by three stretches of amino acids from the correspondingpositions of the kringle 2 domain of t-PA (M²⁸-K³³, D⁵⁵-D⁵⁷and N⁶⁷-W⁷²). These changes resulted in the creation of thelysyl-binding site consensus of the kringle 2 domain (K³³, D⁵⁵,D⁵⁷, W⁶² and W⁷²) in the u-PA kringle. However, the resultingu-PA mutant did not interact with lysyl-Sepharose, nor did itdisplay fibrin-enhanced plasminogen activation in the presenceof soluble fibrin mimic. When the kringle domain of u-PA wasreplaced by the kringle 2 domain of t-PA, similar results wereobtained. The hybrid protein hardly interacted with lysyl-Sepharoseand the plasminogen activation was not enhanced in the presenceof fibrin mimic However, the N-terminal fragment isolated fromthis hybrid molecule (consisting of growth factor domain andkringle 2 domain) did interact with lysyl-Sepharose, suggestingthat in the hybrid molecule a functional lysyl-binding siteis present but not operational. Indeed, lysine analogue (e-amino-caproicacid) sensitive binding of isolated t-PA kringle 2 domain tou-PA could be observed. The modified u-PA kringle, the wildtype u-PA kringle and the kringle 2 of the u-PA hybrid werealso placed N-terminal of the protease domain of t-PA. As expected,the t-PA mutant consisting of the kringle 2 domain and the proteasedomain bound to lysyl-Sepharose and showed fibrin-dependentplasminogen activation. Further, the hybrid molecule consistingof the u-PA kringle placed N-terminal of the t-PA protease domaindid not display these features. Introduction of the modifiedu-PA kringle N-terminal of the t-PA protease domain resultedin a very weak interaction with lysyl-Sepharose. Despite thehigh overall similarity in primary structure of the modifiedu-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogenactivation of this hybrid molecule was observed. The above-mentionedresults question the concept that the structural auto-nomousdomains within hybrid plasminogen activators t-PA and u-PA functionas autonomous domains and suggest that interactions betweenthe kringle and the protease domain in hybrid molecules stronglyinfluences their functional features     

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

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

High-level bacterial expression, purification and characterization of human calreticulin

To investigate its cellular function and role in autoimmunedisease pathogenesis, we have bacterlally expressed human calreticulin,a major calcium-binding protein in the endoplasmic reticulumand a human autoantigen. This is the First report describingthe heterologous expression of calreticulin from any source.The recombinant calreticulin constituted {small tilde}32% ofthe soluble Escherichia coli proteins, and was purified to apparenthomogeneity by ion exchange and hydrophobic liquid chromatography.As does the bona fide protein, the recombinant calreticulinbinds calcium and undergoes changes in its conformation uponZn²⁺ binding. We take this as a strong indication that the foldingof the E.coli-expressed calreticulin is very similar, if notidentical, to that of the authentic protein. Moreover, the bacteriallyexpressed calreticulin readily reacted with anti-human and anti-rabbitantibodies, and the anti-recombinant calreticulin antibodiesimmunoreacted with HeLa calreticulin. The availability of thisexpression system will allow us to carry out site-specific anddeletion mutagenesis analysis in structure-function studiesof calreticulin.     

Site-directed mutagenesis of the putative active site residues of 3C proteinase of coxsackievirus B3: evidence of a functional relationship with trypsin-like serine proteinases

Picornavirus 3C proteinases (3C^pro) are cysteine proteinasesbut recent sequence analyses have shown that they are relatedto trypsin-like serine proteinases. Two models of 3C^pro structurehave been presented. Both models indicate that residues His40and Cysl47 are members of the catalytic triad but the modelsdiffer in the designation of the third member of the catalytictriad, which is assigned as either Glu71 or Asp85. To test theimportance of these four residues in the catalytic activityof 3C^pro of coxsackievirus B3, a member of the enterovirus subgroupof the picornavirus family, single amino acid substitutionswere introduced at each of the four sites. All of these mutationsresulted in the reduction or inactivation of autocatalytic cleavageof the 3C precursor protein expressed in Escherichia coli, suggestingthat all of these residues are essential for the proteolyticreaction. The substitution of Cysl47 with Ala abolished 3C^proactivity while the mutant in which Cysl47 was replaced withSer retained reduced proteolytic activity both in cis and intrans. Our results strongly support the proposal that Cysl47of 3C^pro functions as a nucleophile analogous to Serl95 of trypsin-likeserine proteinases.     

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering

Recognition by ribonuclease T₁ of guanine bases via multidentatehydrogen bonding and stacking interactions appears to be mediatedmainly by a short peptide segment formed by one stretch of aheptapeptide, Tyr⁴²-Asn⁴³-Asn⁴⁴-Tyr⁴⁵-Gly⁴⁶-Gly⁴⁷-Phe⁴⁸. Thesegment displays a unique folding of the polypeptide chain—consistingof a reverse turn, Asn⁴⁴-Tyr⁴⁵-Glu⁴⁶-Gly⁴⁷, stabilized by ahydrogen-bond network involving the side chain of Asn⁴⁴, themain-chain atoms of Asn⁴⁴, Gly⁴⁷ and Phe⁴⁸ and one water molecule.The segment is connected to the C terminus of a ß-strandand expands into a loop region between Asn⁴³ and Ser⁵⁴. Lowvalues for the crystallographic thermal parameters of the segmentindicate that the structure has a rigidity comparable to thatof a ß-pleated sheet. Replacement of Asn⁴⁴ with alanineleads to a far lower enzymatic activity and demonstrates thatthe side chain of Asn⁴⁴ plays a key role in polypeptide foldingin addition to a role in maintaining the segment structure.Substitution of Asn⁴³ by alanine to remove a weak hydrogen bondto the guanine base destabilized the transition state of thecomplex by 6.3 kJ/mol at 37°C. In contrast, mutation ofGlu⁴⁶ to alanine to remove a strong hydrogen bond to the guaninebase caused a destabilization of the complex by 14.0 kJ/mol.A double-mutant enzyme with substitutions of Asn⁴³ by a histidineand Asn⁴⁴ by an aspartic acid, to reproduce the natural substitutionsfound in ribonuclease M_s, showed an activity and base specificitysimilar to that of the wild-type ribonuclease M_s. The segmenttherefore appears to be well conserved in several fungal ribonucleases.     

Altering the association properties of insulin by amino acid replacement

The importance of Pro^B28 and Lys^B29 on the self-associationof insulin was established by systematically truncating theC terminus of the B chain. The relationship between structureand association was further explored by making numerous aminoacid replacements at B²⁸ and B²⁹ Association was studied bycircular dichroism, size-exclusion chromatography and ultracentrifugation.Our results show that the location of a prolyl residue at B²⁸is critical for high-affinity self-association. Removal of Pro^B28in a series of C-terminal truncated insulins, or amino acidreplacement of Pro²⁸, greatly reduced association. The largestdisruption to association was achieved by replacing Lys^B29 withPro and varying the amino acid at B²⁸ Several of the analogswere predominantly monomers in solutions up to 3 mg/ml. Theseamino acid substitutions decreased association by primarilydisrupting the formation of dimers. Such amino acid substitutionsalso substantially reduced the Zn-induced insulin hexamer formation.The formation of monomeric insulins through amino acid replacementswas accompanied by conformational changes that may be the causefor decreased association. It is demonstrated that self-associationof insulin can be drastically altered by substitution of oneor two key amino acids.     

Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants

We have constructed an expression vector that leads to secretionof the whole Fc of human immunoglobulin E (hIgE-Fc) from mammaliancells at levels up to 100 mg/l of culture. Two surface glycosylationsites at Asn265 and Asn371 have been changed to glutamine, toobtain a more homogeneous preparation of hIgE-Fc for structuralstudies. Comparison of wild-type and mutant products revealedthat Asn371 is rarely glycosylated in Chinese hamster ovarycells. Both the double mutant and wild-type hIgEFc bind to thehigh-affinity IgE receptor, FcRI, with about the same affinityas myeloma IgE (K_a in the range 10¹⁰–10¹¹ M^–1),and were able to sensitize isolated human basophils for anti-IgEtriggering of histamine release. However, only the double mutanthIgE-Fc approached the affinity of myeloma IgE for the low-affinityreceptor, FcRII (K_a = 7.3x10⁷ M^–1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (K_a = 4.1x10⁶M^–1).     

Bacterial secretion of the Fab fragment of a mouse monoclonal IgM that reacts with IgG variable regions

In this report, we describe the expression system that enabledus to produce in Escherichia coli the Fab fragment of a mouseIgM that has previously been shown to inhibit the binding ofIgG to autoantigens by interacting with their variable regions.In our system, both light chain and heavy chain fragments wereput under the control of the malE promoter. The light chainwas fused to the MalE signal sequence, while the heavy chainvariable and first constant region were fused to the alkalinephosphatase signal sequence. In this system, after inductionof the promoter with maltose, the Fab fragment could be detectedin a periplasmic extract of the bacteria by Western blottingand also by ELISA. This Fab fragment was purified on a goatanti-mouse immunoglobulin immunoadsorbent and biotinylated.The Fab fragment produced by E.coli reacted with the trinitrophenyl(TNP) hapten and F(ab')₂ fragments of mouse IgG and these reactivitiescould be specifically inhibited by the corresponding solubleantigens. The dissociation constants of this Fab were 1.65 x10^–6 M for TNP and 5 x 10^–6 M for IgG F(ab')₂ fragments,indicating that the affinity of the Fab fragment compared withthat of the whole IgM molecule was similar for TNP but was lowerfor IgG F(ab')₂ fragments     

Reaction mechanism of trypsin-catalysed semisynthesis of human insulin studied by fast atom bombardment mass spectrometry

The production of semisynthetic human insulin for therapeuticpurposes is of considerable importance. During trypsin-catalysedtransformation of pig insulin into an ester of insulin of humansequence, the alanyl residue at position B³⁰ is removed andreplaced with an esterified residue of threonine. We have carriedout this transformation in a medium enriched in ¹⁸OH₂ and studiedthe product by MS. In contrast to a previous report, we findthat incorporation of label into the B²⁹–B³⁰ peptide bondoccurs during the transformation with threonine methyl esterin aqueous N, N-dimethylacetamide. Quantitative data are presentedand the implications of these findings are discussed.     

The semisynthesis of octadeutero-PheB1-octadeutero-ValB2]-procine insulin and its characterization by mass spectrometry

Insulin analogues labelled with stable isotopes (e.g. deuterium,¹⁸O, ¹⁵N, etc.) are authentic (the native structure is rigorouslymaintained), non-radioactive (preferred for injection into man)and can easily be distinguished from endogenous insulin by massspectrometry by virtue of their molecular masses. Appropriatecombinations of amino-protecting groups (methylsulphonylethyloxycarbonyland t-butoxy carbonyl), Edman degradation and chemical couplingwere used to produce [octadeutero-Phe^B1]-porcine insulin and[octadeutero-Phe^B1-octadeutero-Val^B2]-porcine insulin. The analogueswere characterized by electrospray ionization mass spectrometry.Standard mixtures of labelled and unlabelled insulins were successfullystudied by mass spectrometry. Isotope dilution mass spectrometrycould therefore provide a useful direct measure of insulin undertrue physiological conditions, without many of the drawbacksof existing methods. In this regard, the analogue with 16 deuteriumswas more suitable than the octadeuterated analogue, since thegreater mass difference between the labelled and unlabelledforms enabled a lower mass spectrometric resolution to be used,resulting in higher sensitivity     

Stabilization of lysozyme by the introduction of Gly-Pro sequence

Three mutant lysozymes where the Asp¹⁰¹ – Gly¹⁰² sequenceof lysozyme was converted to Asp¹⁰¹–Pro¹⁰², Gly¹⁰¹–Pro102and Pro¹⁰¹–Gly¹⁰² were prepared to investigate the effectof proline residues on the stabilization of proteins. The freeenergy changes of lysozymes for the unfolding in aqueous solutionat pH 5.5 and 35°C were 10.0, 10.1, 11.0 and 7.7 kcal/molfor wild type, Asp¹⁰¹Pro¹⁰², Gly¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰² lysozymerespectively. When the energy level in the unfolded state ofwild type lysozyme was fixed at a standard level, the energylevels in the folded state of Asp¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰²lysozymes were found to be higher than that of wild type lysozymeon the basis of G_D(H₂O) and entropy losses of their polypeptidechains in the unfolded state. The presence of some strain inthe folded state of these lysozymes was supported by both thecalculation of conformational energy for a trans-L-prolyl residue[Schimmel, P.R. and Flory,P.J. (1968) J. Mol. Biol, 34, 105–120] and the analysis of structures of energy-minimizedmutant lysozymes. Therefore, it is concluded that the formationof the Gly-Pro sequence is effective in avoiding possible strainin the folded state of a protein caused by the introductionof proline residue(s).     

Site-directed mutagenesis of pseudoazurin from Alcatigenes faecalis S-6; Pro80Ala mutant exhibits marked increase in reduction potential

Pseudoazurin (a blue copper protein or cupredoxin) of a denitrifyingbacterium Alcaligenes faecalis S-6 is a direct electron carrierfor a Cu-containing nitrite reductase (NIR) of the same organism.Site-directed mutagenesis of the pseudoazurin was carried outusing an Escherichia coli expression system. Replacement ofTyr74 by Phe to remove an internal hydrogen bond in the ß-barrelcaused a slight decrease in heat stability as well as a requirementfor a higher concentration of Cu²⁺ for production in the E.colihost. Exchange of Ala for Pro80 adjacent to His81, one of thefour ligands binding a type I Cu atom, caused a marked increasein reduction potential by 139 mV without change in the opticalabsorption spectrum. The ability of the pseudoazurin to transferelectrons to NIR was markedly diminished but the apparent K_mof NIR for pseudoazurin was not affected by the mutation. X-raydiffraction data collected on the oxidized and reduced formsof the Pro80Ala mutant show that a water molecule occupies thepocket created by the absent side chain. This observation suggeststhat the increase in reduction potential may be caused due tothe increased solvent accessibility to the Cu atom. The electrondensity difference maps on these structures (at 2.0 Å)show that this water moves during the change in oxidation state,and that there are small, but localized, conformational changes>6.5 Å from the copper site, as well as movement ofboth the Cu²⁺ and the cysteinate sulfur.     

Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity

The expression of immunoglobulin heavy and light chain variableregions in the cytoplasm of Escherichia coli and formation ofa functional heterodimer has been demonstrated. Variable domainsequences were taken from the heavy and light chain cDNAs ofthe monoclonal antibody Gloop 2 and engineered for expressionin a dual origin expression vector. The engineered genes vhg2and vlg2 were separately subcloned into the vector, creatingtwo expression plasmids. Expression of the heavy and light chainvariable region genes (encoding 116 and 109 amino adds respectively)was investigated in eight E.coli strains; the polypeptides wererapidly degraded in a host strain optimized for expression andin E.coli strains deficient in the major protease La (lon^-).Accumulation was permitted in severely protease-deficient E.colihaving a defective heat-shock response. A lon^- mutation in thisgenetic background permitted even higher accumulation. Expressionlevels were 7 and 1% of total bacterial protein for light andheavy chain variable regions respectively. Expression of theheavy chain variable region gene was increased by includinga longer Shine–Dalgarno sequence. Similar constructionsin the light chain vector had no effect on expression levels.The insoluble variable region polypeptides were reconstitutedinto a heterodimer possessing the full antigen binding characteristicsof both the parent monoclonal antibody and its Fab fragment.     

Molecular modelling of xylose isomerase catalysis: the role of electrostatics and charge transfer to metals

The two main steps of the mechanism of xylose-xylulose conversioncatalysed by D-xylose isomerase, the ring opening of xyloseand the isomerization of the opened product by hydride transfer,were investigated by molecular mechanical and molecular orbitaltechniques. The activation energies calculated for these reactionsclearly showed that hydrogen transfer is the rate-determiningstep of the enzymatic isomerization and that Mg²⁺ ions activatewhereas Zn²⁺ ions inhibit the reaction, in agreement with theexperiments. The remarkable differences between the net chargesof these ions found by molecular orbital calculations and theinspection of the protein electrostatic potential around thereaction intermediates indicate that the main role of bivalentmetal ions should be the electrostatic stabilization of thesubstrate transition states. In order to propose a more detailedmechanism, an attempt was made to clarify the effects of nearbyresidues (e.g. His54, Asp57, Lysl83, Asp257) in the reaction.Different isomerization mechanisms, such as through an enediolintermediate, were examined and could be excluded, in additionto the charge-relay mechanism during the ring opening.     

Expression of recombinant {alpha}Ains-crystallin and not {alpha}A-crystallin inhibits bacterial growth

A-Crystallin and A^ins-crystallin are derived from the A-crystallingene via alternative splicing. They are identical except forthe presence of a polypeptide, 23 amino acids long, encodedby the ‘insert’ exon. Evolutionary logic would suggestthat the insertion of a 23 amino acid peptide in the middleof A-crystallin, a protein evolving more slowly than eitherhistone H1, cytochrome c or hemoglobin, would lead to appreciablestructural and functional changes. However, based on physico-chemicalstudies, it is presently believed that A-crystallin and A^ins-crystallinare functionally equivalent and that the presence of the ‘insert’peptide in A^Ins-crystallin is inconsequential. We report herethat the independent expression of recombinant A^Ins-crystallin,and not A-crystallin, inhibits growth of the bacterial host.These observations were confirmed in co-expression experiments,wherein both the proteins were expressed in the same cell. Interestingly,growth inhibition is reversible. Importantly, the data demonstratethat it is catalytic amounts and not the gross accumulationof A^Ins-crystalline which causes growth inhibition. Given theprior knowledge that A-crystallin and A^Ins-crystallin differby a peptide of 23 amino acids, these data suggest that the‘insert peptide’ in A^Ins-crystallin imparts propertieson this protein that are different from A-crystallin.     

Synthesis of a squash-type protease inhibitor by gene engineering and effects of replacements of conserved hydrophobic amino acid residues on its inhibitory activity

Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of thesquash-type protease inhibitor family, is composed of 29 aminoacids and shows strong inhibition of trypsin by its compactstructure. To study the structure–function relationshipof this inhibitor using protein engineering methods, we constructedan expression system for CMTI-I as a fused protein with porcineadenylate kinase (ADK). A Met residue was introduced into thejunction of ADK and CMTI-I to cleave the fusion protein withCNBr, whereas a Met at position 8 of authentic CMTI-I was replacedby Leu. Escherichia coli JM109 transformed with the constructedplasmid expressed the fused protein as an inclusion body. Aftercleavage of the expressed protein with CNBr, fully reduced speciesof CMTI-I were purified by reversed-phase HPLC and then oxidizedwith air by shaking. For efficient refolding of CMTI-I, we used50 mM NH₄HCO₃ (pH 7.8) containing 0.1% PEG 6000 at higher proteinconcentration. Strong inhibitory activity toward trypsin wasdetected only in the first of three HPLC peaks. The inhibitorconstant of CMTI-I thus obtained, in which Met8 was replacedby Leu, was 1.4x10-¹⁰ M. The effect of replacement of Met withLeu at position 8 was shown to be small by comparison of theinhibitor constant of authentic CMTI-III bearing Lys at position9 (8.9x10-¹¹ M) with that of its mutant bearing Leu at position8 and Lys at position 9 (1.8x10-¹⁰ M). To investigate the roleof the well conserved hydrophobic residues of CMTI-I in itsinteraction with trypsin, CMTI-I mutants in which one or allof the four hydrophobic residues were replaced by Ala were prepared.The inhibitor constants of these mutants indicated that thosewith single replacements were 5–40 times less effectiveas trypsin inhibitors and that the quadruple mutant was –450times less effective, suggesting that the hydrophobic residuesin CMTI-I contribute to its tight binding with trypsin. However,each mutant was not converted to a temporary inhibitor.