Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin

A gene for expression of horse heart myoglobin in Escherichiacoli has been constructed in one step from long synthetic oligonucleotides.The synthetic gene contains an efficient translation initiationsignal and used codons that are commonly found in E.coli. Uniquerestriction sites are placed throughout the gene. It has beeninserted in a phagemid vector and is expressed from the lacpromoter in E.coli at high efficiency, the soluble heme proteinrepresenting 10% of soluble protein. Two versions of horse heartmyoglobin were produced with aspartic acid or asparagine atresidue 122. Comparison of chromatographic mobilities of thesetwo proteins with authentic horse heart myoglobin identifiedaspartic acid as the correct residue 122. The availability ofthis gene, which is designed to facilitate oligonucleotide mutagenesisor cassette mutagenesis, will allow systematic structure—functionanalysis of horse heart myoglobin.     

Expression, purification and crystallization of penicillin G acylase from Escherichia coli ATCC 11105

The penicillin acylase gene (pac) from Escherichia coli ATCC11105 was cloned into pUC 9 and the resulting vector (pUPA-9),when transformed into E.coli strain 5K, allowed the constitutiveoverproduction of mature penicillin acylase when grown at 28°C.The enzyme ws purified from the periplasmic fraction of E.colipUPA-9 by hydrophobic interaction chromatography and anion exchange.Crystals of penicillin acylase were grown in batch using polyethyleneglycol 8000 as a precipitant. The crystals (space group P1)diffracted to beyond 2.3 Å.     

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.     

Automated protein crystallization and a new crystal form of a subtilisin:eglin complex

A procedure is described for automating labour-intensive stepsof the ‘hanging drop’ protein crystallization method.An automatic sample changer is employed to fill the wells ina multi-well plate so that concentration gradients in variouscomponents are obtained. The sample changer is also used forpreparing droplets on a second multi-well plate. Subse quently,this second plate is manually turned around and placed on topof the first multi-well plate such that a large number of chamberswith different conditions is obtained simultaneously. Duringinitial trials a new crystal form of a subtilisin:eglin complexwas obtained. The crystals have space group P2₁ contain twoenzyme inhibitor complexes per asymmetric unit and diffractbeyond 2.2 Å.     

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase -subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of k_cat; however, K_m values for both peptide and FPPincreased 2–3-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity.     

A robust and efficient automated docking algorithm for molecular recognition

A completely automated method is described for determining themost likely mode of binding of two (macro)moJecules from theknowledge of their three-dimensional structures alone. The methodis based on well-known graph theoretical techniques and hasbeen used successfully to determine and rationalize the bindingof a number of known macromolecular complexes. In this articlewe present results for a special case of the general molecularrecognition problem—given the information concerning theparticular atoms involved in the binding for one of the molecules,the algorithm can correctly identify the corresponding (contacting)atoms of the other molecule. The approach used can be easilyextended to the general molecular recognition problem and requiresthe extraction of maximal common subgraphs. In these studiesthe docking of the macromolecules was achieved without the aidof computer graphics or other visual aids. The algorithm hasbeen used to determine the correct mode of binding of a proteinantigen to an antibody in 100 min on a DEC micro VAX 3600     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli

For the first time the pro-form of a recombinant cysteine proteinasehas been expressed at a high level in Escherichia coli. Thisinactive precursor can subsequently be processed to yield activeenzyme. Sufficient protein can be produced using this systemfor X-ray crystallographic structure studies of engineered proteinases.A cDNA clone encoding propapain, a precursor of the papaya proteinase,papain, was expressed in E.coli using a T7 polymerase expressionsystem. Insoluble recombinant protein was solubilized in 6 Mguanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6.A protein-glutathione mixed disulphide was formed by dilutioninto oxidized glutathione and 6 M GuHCl, also at pH 8.6. Finalrefolding and disulphide bond formation was induced by dilutioninto 3 mM cysteine at pH 8.6. Renatured propapain was processedto active papain at pH 4.0 in the presence of excess cysteine.Final processing could be inhibited by the specific cysteineproteinase inhibitors E64 and leupeptin, but not by pepstatin,PMSF or EDTA. This indicates that final processing was due toa cysteine proteinase and suggests that an autocatalytic eventis required for papain maturation.     

Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.     

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.     

The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides

The Ecballium elaterium trypsin inhibitor II (EETI-II), a memberof the squash family of protease inhibitors, is composed of28 amino acid residues and is a potent inhibitor of trypsin.Its compact structure is defined by a triple-stranded antiparallelß-sheet, which is held together by three intramoleculardisulfide bonds forming a cystine knot. In order to explorethe potential of the EETI-II peptide to serve as a structuralscaffold for the presentation of randomized oligopeptides, weconstructed two EETI-II derivatives, where the six-residue inhibitorloop was replaced by a 13-residue epitope of Sendai virus L-proteinand by a 17-residue epitope from human bone Gla-protein. EETI-IIand derived variants were produced via fusion to maltose bindingprotein MalE. By secretion of the fusion into the periplasmicspace, fully oxidized and correctly folded EETI-II was obtainedin high yield. EETI-II and derived variants could be presentedon the Escherichia coli outer membrane by fusion to truncatedLpp'–OmpA', which comprises the first nine residues ofmature lipoprotein plus the membrane spanning ß-strandfrom residues 46–66 of OmpA protein. Gene expression wasunder control of the strong and tightly regulated tetA promoter/operator.Cell viability was found to be drastically reduced by high levelexpression of Lpp'–OmpA'–EETI-II fusion protein.To restore cell viability, net accumulation of fusion proteinin the outer membrane was reduced to a tolerable level by introductionof an amber codon at position 9 of the lpp' sequence and utilizingan amber suppressor strain as expression host. Cells expressingEETI-II variants containing an epitope were shown to be surfacelabeled with the respective monoclonal antibody by indirectimmunofluorescence corroborating the cell surface exposure ofthe epitope sequences embedded in the EETI-II cystine knot scaffold.Cells displaying a particular epitope sequence could be enriched10⁷-fold by combining magnetic cell sorting with fluorescence-activatedcell sorting. These results demonstrate that E.coli cell surfacedisplay of conformationally constrained peptides tethered tothe EETI-II cystine knot scaffold has the potential to becomean effective technique for the rapid isolation of small peptidemolecules from combinatorial libraries that bind with high affinityto acceptor molecules.     

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.     

Engineering of the Escherichia coli Im7 immunity protein as a loop display scaffold

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.     

Generating molecular diversity by homologous recombination in Escherichia coli

We explored the use of recE-mediated homologous recombination to generate molecular diversity in Escherichia coli. Two homologous genes were placed on different phagemid vectors each comprising multiple EcoRI restriction sites and overlapping N- and C-terminal portions of beta-lactamase. By co-infection of these phage into RecE+ EcoRI+ E.coli, we were able to introduce double-strand breaks into these vectors, allowing efficient homologous recombination (in up to 10% of bacteria) by the recE pathway and selection of the recombinants by resistance to ampicillin. Recombination gave single crossovers; these were more frequent near the EcoRI sites and the recombination frequency increased with the target length and degree of homology. The system was used to create a large combinatorial chicken antibody library (10(10)) for display on filamentous phage and to isolate several antibody fragments with binding affinities in the 10-100 nM range.     

Molecular surface recognition by a computer vision-based technique

Correct docking of a ligand onto a receptor surface is a complexproblem, involving geometry and chemistry. Geometrically acceptablesolutions require close contact between corresponding patchesof surfaces of the receptor and of the ligand and no overlapbetween the van der Waals spheres of the remainder of the receptorand ligand atoms. In the quest for favorable chemical interactions,the next step involves minimization of the energy between thedocked molecules. This work addresses the geometrical aspectof the problem. It is assumed that we have the atomic coordinatesof each of the molecules. In principle, since optimally matchingsurfaces are sought, the entire conformational space needs tobe considered. As the number of atoms residing on molecularsurfaces can be several hundred, sampling of all rotations andtranslations of every patch of a surface of one molecule withrespect to the other can reach immense proportions. The problemwe are faced with here is reminiscent of object recognitionproblems in computer vision. Here we borrow and adapt the geometrichashing paradigm developed in computer vision to a central problemin molecular biology. Using an indexing approach based on atransformation invariant representation, the algorithm efficientlyscans groups of surface dots (or atoms) and detects optimallymatched surfaces. Potential solutions displaying receptor-ligandatomic overlaps are discarded. Our technique has been appliedsuccessfully to seven cases involving docking of small molecules,where the structures of the receptor-ligand complexes are availablein the crystallo-graphk database and to three cases where thereceptors and ligands have been crystallized separately. Intwo of these three latter tests, the correct transformationshave been obtained.     

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.     

Molecular dynamics simulation of fungal cellulose-binding domains: differences in molecular rigidity but a preserved cellulose binding surface

A total of 23 fungal cellulose-binding domain (CBD) sequenceswere aligned. Structural models of the cellulosebinding domainof an exoglucanase (CBHII) and of three endoglucanases (EGI,EGII and EGV) from Trichoderma reesei cellulases were homologymodelled based on the NMR structure of the fungal cellobiohydrolaseCBHI, from the same organism. The completed models and the knownstructure of the CBHI cellulose-binding domain were refinedby molecular dynamics simulations in water. All four modelswere found to be very similar to the structure of the CBHI cellulose-bindingdomain and sequence comparison indicated that in general thethree-dimensional structures of fungal cellulose-binding domainsare very similar. In all the CBDs studied, two disulphide bridgesapparently stabilize the polypeptide fold. From the models,an additional disulphide bridge was predicted in EGI and CBHII,and in eight further CBDs from other organisms. Three highlyconserved aromatic residues on the hydrophilic side of the wedgemake this surface flat This surface is expected to make contactwith the substrate. Three invariant amino acids, Gln7, Asn29and Gln34, on this flat face are in suitable positions for hydrogenbonding with the cellulose surface. Analysis of the differencesin the protein surface properties indicated that the endoglucanasestend to be more hydrophilic than the exoglucanases. The largeststructural variation was found around positions 12-16. The fungalCBD sequences are discussed in relation to variations in functionand pH dependence. Comparison of the modelled structures withexperimental binding data for the CBHI and EGI allowed the formulationof a qualitative relationship to cellulose affinity. This relationshipwas used to predict the cellulose affinities for 21 CBDs.     

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

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

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increasedor decreased to alter the electrostatic interaction betweenenzyme and substrate in the lytic action of human lysozyme usingsite-directed mutagenesis. The amino acid substitutions accompanyingeither the addition or the removal of two units of positivecharge have shifted the optimal ionic strength (NaCl concentrationin 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticuscell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively.In addition to the change in ionic strength–activity profile,the pH–activity profile and the effect of a polycationicelectrolyte, poly-L-Lys-HCl, on the lytic activity were significantlychanged. Owing to the shifts in both ionic strength profilesand pH profiles the Arg⁷⁴/Arg¹²⁶ mutant has become a bettercatalyst than wild-type enzyme under the conditions of highionic strength and high pH, and the Gln⁴¹/Ser¹⁰¹ mutant hasbecome a better catalyst under the conditions of low ionic strengthand low pH.     

High-yield expression in Escherichia coli of soluble human alpha-hemoglobin complexed with its molecular chaperone

The alpha-subunits of human hemoglobin (Hb) have been more difficult to express than beta-chains owing to the high instability of alpha-chains. Here, we describe the production in Escherichia coli of a soluble recombinant alpha-Hb with human alpha-hemoglobin-stabilizing protein (AHSP), its molecular chaperone. To succeed in this expression, we have constructed a vector pGEX-alpha-AHSP which contains two cassettes arranged in tandem in the same orientation permitting to express alpha-hemoglobin and human AHSP. While the GST-alpha-Hb alone was expressed in E.coli as insoluble protein, even after adding lysate containing recombinant AHSP, the expression vector pGEX-alpha-AHSP permits the co-expression of soluble GST-alpha-Hb and GST-AHSP. The alpha-Hb, produced at a high yield of 12 to 20 mg per liter of culture, was then purified as a complex with its chaperone. Biochemical and biophysical properties of recombinant AHSP/recombinant alpha-Hb complex were similar to those of recombinant AHSP/native alpha-Hb complex as assessed by UV/visible and CO or O(2) binding properties. This co-expression technique can be use to study the interaction between a molecular chaperone and its target protein and, more generally, this system would be particularly interesting for the study of partner proteins when one or both proteins are individually unstable.     

Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli

We have developed a high-level production system for the C-terminaldomain of secretory leukoprotease inhibitor (SLPI) to investigateits pharmacological activities. A gene for the C-terminal domainof SLPI, (Asn55-AlalO7)SLPI, was constructed from chemicallysynthesized deoxyoligonucleotides. It was fused to a gene forthe N-terminal portion of human growth hormone via a DNA sequenceencoding Leu-Val-Pro-Arg, which can he cleaved by thrombin.The fused gene was expressed in Escherichia coli under the controlof a trp promoter, and the fusion protein was obtained as aninclusion body. After sulfonation of the cysteine residues,the sulfonated fusion protein was cleaved at the desired siteby thrombin. Sulfonated (Asn55-Ala107)SLPI was refolded in Trisbuffer containing reduced and oxidized glutathione. The resulting(Asn55-Ala107) SLPI was purified by cation-exchange chromatographyand reverse-phase high performance liquid chromatography. Thefinal yield was 50 mg/l culture. (Asn55-Ala107)SLPI was as activeagainst elastase as, but had less trypsin inhibitory activitythan, native SLPI. This system is suitable for the large-scaleproduction of the C-terminal domain of SLPI, which is an elastase-specificinhibitor.