Secretory Expression System Research Articles

Nasal immunization with E. coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion–expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract).

Yeast secretory expression of insulin precursors

Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretory expression system in S. cerevisiae and its subsequent optimisation is described. Expression of proinsulin in S. cerevisiae does not result in efficient secretion of proinsulin or insulin. However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine(B30) as a fusion protein with the S. cerevisiae alpha-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g. AAK), or by direct fusion of lysine(B29) to glycine(A1), results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S. cerevisiae and Pichia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield.

Production of granulocyte-macrophage colony stimulating factor (GM-CSF) by high cell density fermentation of secretory recombination Escherichia Coli

High cell density fermentation production of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) was studied in an Escherichia coli secretory expression system. By using glycerol instead of glucose as a carbon source and feeding in a specific fed-batch mode and by controlling the temperature at about 30°C and maintaining the dissolved oxygen level at 20∼25% saturation, cell density increased from 30 to 91.8 g l −1, the production of recombinant GM-CSF remained high and the fermentation time was reduced from 45 to 30 h. Recombination GM-CSF was purified by hydrophobic column-molecular sieve-ion exchange column chromatography. The expression of recombinant GM-CSF was not affected by fermentation methods, the production of GM-CSF reached 28.04% (about 1.68 g l −1) and 27.51% (about 1.64 g l −1) of total somatic proteins for high cell density and normal fermentation, respectively. The corresponding value for specific activity of GM-CSF was 2.6×10 7, and 2.4×10 7, respectively.

Secretory expression of human albumin domains in Saccharomyces cerevisiae and their binding of myristic acid and an acylated insulin analogue.

Albumin is organized in three homologous domains formed by double loops stabilized by disulfide bonds. Utilizing a secretory expression system based on a synthetic secretory prepro-leader, the three human serum albumin domains were expressed in the yeast Saccharomyces cerevisiae. Human serum albumin domains I and III were efficiently expressed and secreted, indicating that these domains can form independent structural units capable of folding into stable tertiary structures. In contrast, albumin domain II was not secreted and disappeared early in the secretory pathway. Human serum albumin has the ability to bind a large number of small molecule ligands, including fatty acids, presumably due to its structure and structural flexibility. Purified albumin domain III bound myristic acid, whereas purified albumin domain I did not bind myristic acid. A new soluble long-acting insulin an alogue acylated with myristic acid (Markussen J., et al., Diabetologia 39, 281-288, 1996) bound to domain III and bound markedly more weakly to domain I.

Expression and secretion of the S2 subunit of pertussis toxin in Bacillus brevis

We have been trying to develop a mass production system for each of the subunits (S2, S3, S4, S5) of the pertussis toxin B oligomer (PTB) by using a Bacillus brevis-pNU212 system. In consequence a moderately efficient expression-secretion system for S2 was constructed by fusing the mature S2 gene from Bordetella pertussis Tohama with the signal-peptide coding region of pNU212 and by introducing the plasmid pNU212-S2 into B. brevis HPD31 by electroporation. The clone producing S2 secreted about 70 mg of recombinant S2 (rS2) per liter of PY-erythromycin medium after 5 days incubation at 37°C. The rS2 purified by an ammonium sulfate fractionation at 30–50% saturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was identical to the native S2 in respect to the molecular weight determined by SDS-PAGE and the amino terminal amino acid sequence. The nucleotide sequence of the S2 gene in B. pertussis Tohama inserted into pNU212 was identical with that of the S2 gene in other virulent B. pertussis strains except that an adenine at position 52 of the latter was replaced by a guanine in the former, causing an amino acid substitution (glycine in the former for serine in the latter) at position 18.

Efficient expression, processing and secretion of a biologically active mammalian protein by Vibrio cholerae

The use of Vibrio cholerae as a secretory expression system for the expression of a mammalian protein, namely human growth hormone, under the control of the heat labile enterotoxin chain B signal sequence is reported. The protein is efficiently expressed and processed. The mature protein is exported to the periplasm after which it is secreted to the extracellular milieu. The expressed and secreted hGH actively binds to its receptor as established by its receptor binding activity. The biological activity of the protein is demonstrated in vitro in a Nb2 proliferation assay.

Institutional Policies and Guidelines on Adjuvants and Antibody Production.

Pollard and J. M. Walker, eds. Clifton, N.J.: Humana Press. Reichmann, L., M. Clark, H. Waldmann, and G. Winter. 1988. Reshaping human antibodies for therapy. Nature 332:323-327. Reiter, Y., U. Brinkmann, K. O. Webber, SH. Jung, B. Lee, and I. Pastan. 1994. Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: Improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv. Protein Eng. 7(5):667-704. Roberts, S., J. C. Cheetham, and A. R. Rees. 1987. Generation of an antibody with enhanced affinity and specificity for its antigen by protein engineering. Nature 328:731-734. Roberts, V. A., E. D. Getzoff, and J. A. Tainer. 1993. Structural basis of antigenic cross-reactivity. Pp. 31-53 in Structure of Antigens, Vol. 2. M. H. V. van Regenmortel, ed. Boca Raton, Fla.: CRC Press. Roberts, V. A., J. Stewart, S. J. Benkovic, and E. D. Getzoff. 1994. Catalytic antibody model and mutagenesis implicate arginine in transitionstate stabilization. J. Mol. Biol. 235:1098-1116. Skerra, A., M. L. Dreher, and G. Winter. 1991. Filter screening of antibody Fab fragments secreted from individual bacterial colonies: Specific detection of antigen binding with a two-membrane system. Anal. Biochem. 196:151-155. Soderlund, E., A. C. Simonsson, and C. A. K. Borrebaeck. 1992. Phage display technology in antibody engineering: Design of phagemid vectors and in vitro maturation systems. Immunol. Revs. 130:109-124. Taki, S., M. Meiering, and K. Rajewsky. 1993. Targeted insertion of a variable region gene into the immunoglobulin heavy chain locus. Science 262:1268-1270. Tsumoto, K., Y. Nakaoki, Y. Ueda, K. Ogasahara, K. Yutani, K. Watanabe, and I. Kumagai. 1994. Effect of the order of antibody variable regions on the expression of the single-chain HyHELlO Fv fragment in E. coli and the thermodynamic analysis of its antigen-binding properties. Biochem. Biophys. Res. Comm. 201(2):546-551. Waterhouse, P., A. D. Griffiths, K. S. Johnson, and G. Winter. 1993. Combinatorial infection and in vivo recombination: A strategy for making large phage antibody repertoires. Nucl. Acids Res. 21(9):2265-2266. Whitlow, M., and D. Filpula. 1991. Single-chain Fv proteins and their fusion proteins. METHODS: A Companion to Methods in Enzymology 2(2):97-105. Whitlow, M., B. A. Bell, SL. Feng, D. Filpula, K. D. Hardman, S. L. Hubert, M. L. Rollence, J. F. Wood, M. E. Schott, D. E. Milenic, T. Yokota, and J. Schlom. 1993. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Eng. 6(8):989-995. Wilson, I. A., and R. L. Stanfield. 1993. Antibody-antigen interactions. Curr. Opin. Struct. Biol. 3:113-118. Wu, XC , SC. Ng, R. I. Near, and SL. Wong. 1993. Efficient production of a functional single-chain antidigoxin antibody via an engineered Bacillus subtilis expression-secretion system. Bio/Technol. 11:71-76. Zou, YR., H. Gu, and K. Rajewsky. 1993. Generation of a mouse strain that produces immunoglobulin K chains with human constant regions. Science 262:1271-1274.

Contribution to antibody-antigen interaction of structurally perturbed antigenic residues upon antibody binding.

For elucidating the contribution of structurally perturbed antigenic residues upon antibody binding to antigen-antibody interaction, the interaction between hen egg white lysozyme (HEL) and HyHEL10 Fv fragment, which is one of several monoclonal antibodies against HEL and structurally well defined (Padlan, E.A., Silverton, E. W., Sheriff, S., Cohen, G. H., Smith-Gill, S. J., and Davies, D. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5938-5942), was investigated. Asp-101 and Trp-62 of HEL, whose conformations are perturbed by the binding of antibody HyHEL10 in this interaction, were replaced with Gly, and the resulting interactions were studied by assay of the inhibition of the lysozyme activity with the Fv fragment and by titration calorimetry. The results can be summarized as follows. 1) It was possible to prepare the fully functional Fv fragment of HyHEL10 using a secretory expression system in Escherichia coli. Its inhibition profile for HEL activity was almost indistinguishable from that of HyHEL10 IgG, and the contribution of enthalpy to driving the interaction was shown to be significant. 2) A thermodynamic study of the interaction between the D101G mutant HEL and the Fv fragment revealed that, although the negative enthalpy change was smaller than that for the wild type, the Gibbs energy was almost identical to that of the wild type, which resulted from the smaller entropy loss. 3) Study of the interaction between the W62G mutant HEL and this Fv fragment indicated that the rotation of the Trp-62 indole ring upon binding of the antibody made an enthalpic contribution to antibody-antigen interaction, although Trp-62 of HEL was proposed not to be the direct contact residue in the HyHEL10.HEL complex. 4) From these results, it was confirmed experimentally that structural perturbations of antigenic residues upon antibody binding of antigen would contribute to the gain of enthalpic energy, in spite of partial offset by entropic loss, and to driving the interaction.

Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue

Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases. To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E. coli alkaline phosphatase. E. coli JM105 was transformed with the resulting plasmid and induced with l mM isopropyl-β- d-thiogalactopyranoside (IPTG). The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography. Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site. Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form. Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity.

An Expression System for Secretion and Purification of a Genetically Engineered Thermostable Chimera of Protein A and Alkaline Phosphatase

A chimera between gene segments of Protein A and a mutated alkaline phosphatase (lysine328 mutated to alanine) of Escherichia coli has been constructed. This chimeric gene was cloned in a T7 promoter-based IPTG-inducible expression vector. The chimeric protein was expressed in E. coli and was efficiently secreted into the periplasm, from which it could he easily purified by a combination of ion-exchange and gel permeation chromatography. The purified chimera was found to be thermostable and exhibited both IgG binding and high alkaline phosphatase activity. It was used as a probe in enzyme-linked immunosorbent assays and results indicate that it is a promising substitute for secondary antibodies in enzyme-linked immunoassays.

Heterologous protein secretion directed by a repressible acid phosphatase system of Aspergillus niger

A new expression-secretion system of Aspergillus niger which directs the secretion of heterologous proteins is described. The promoter and signal peptide-encoding region of the phosphate-repressible aphA gene of A. niger, when fused to the coding region of the human interferon α2 (hIFNα2)-encoding gene ( hIFNα2), drives the expression of this gene and the secretion of the hIFNα2 protein. Synthesis of hIFNα2 in either A. niger or A. nidulans transformants carrying these constructs was regulated by inorganic phosphate (P i) present in the medium, so that derepression of heterologous protein expression can be attained by lowering P i concentration.

Efficient Production of a Functional Single-Chain Antidigoxin Antibody via an Engineered Bacillus subtilis Expression-Secretion System

We have applied a Bacillus subtilis expression-secretion system to produce a functional antidigoxin SCA (single-chain antibody consisting of VL-linker-VH) and the individual variable domains of light (VL) and heavy (VH) chains. The secreted antidigoxin SCA can be affinity purified in one step by applying the culture supernatant directly to a ouabain-Sepharose column. N-terminal sequence determination indicated that the protein has the expected N-terminus with the signal peptide properly processed. Affinity and ligand specificity studies demonstrated that the engineered antidigoxin SCA has almost identical properties as those of the parental monoclonal antibody. The use of B. subtilis WB600, an engineered, six-extracellular protease-deficient strain, is vital for the production of antidigoxin SCA in high quality and quantity (5 mg/liter in a shake flask culture). All the secreted SCAs are biologically active. The ability to produce secreted SCAs by the B. subtilis expression system provides a simple and efficient means to analyze the binding properties of engineered antibodies generated through rational design or site-directed mutagenesis.

Synthesis and secretion of an Erwinia chrysanthemi pectate lyase in Saccharomyces cerevisiae regulated by different combinations of bacterial and yeast promoter and signal sequences

Nine different expression-secretion cassettes, comprising novel combinations of yeast and bacterial gene promoters and secretion signal sequences, were constructed and evaluated. A pectate lyase-encoding gene ( pelE) from Erwinia chrysanthemi was inserted between each one of these expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMSl through pAMS9. These YIp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae, and the pectate lyase production was monitored. Transcription initiation signals for pelE expression were derived from the yeast alcohol dehydrogenase ( ADC1 P ), the yeast mating pheromone α-factor ( MFα1 P ) and the Bacillus amyloliquefaciens α-amylase ( AMY P ) gene promoters. The transcription termination signals were derived from the yeast tryptophan synthase gene terminator ( TRP5 T ). Secretion of pectate lyase (PLe) was directed by the signal sequences of the yeast mating pheromone α-factor ( MFα1 S ), B. amyloliquefaciens α-amylase ( AMY S ) and Er. chrysanthemi pectate lyase ( pelE S ). The ADCl P - MFα1 S expression-secretion system proved to be the most efficient control cassette for the expression of pelE and the secretion of PLe in S. cerevisiae.

Promoter constructions for efficient secretion expression in Streptomyces lividans.

Promoters from different Streptomyces genes were cloned in front of the Tendamistat gene from S. tendae, in order to study secretion-expression in S. lividans using a pIJ702 plasmid vector system. Besides the promoters we cloned a transcriptional terminator downstream of the Tendamistat gene to improve transcription efficiency. The promoters we selected were: (1) a synthetic Escherichia coli-like consensus promoter; (2) the aphI promoter of the neomycin resistance gene from S. fradiae; (3) an ermE-up promoter mutant from Saccharopolyspora erythraea; (4) the melC promoter of the tyrosinase operon from Streptomyces antibioticus. In addition, we tested the thiostrepton-inducible tipA promoter from S. lividans in our Tendamistat secretion system. The promoters were cloned upstream of the Tendamistat ribosome binding site in order to conserve the original translation initiation. The Tendamistat secretion mediated by the different promoter constructions above varied dramatically in up to 10 mg/l in the case of the synthetic promoter and the aph promoter, and up to 500 mg/l mediated by the ermE-up promoter. The melC promoter allowed about 200 mg/l Tendamistat secretion and the tipA promoter proved to be inducible from less than 0.5 mg/l up to 40 mg/l of Tendamistat secretion. Based on the amount of secreted Tendamistat and on the analysis of mRNA levels, we conclude that transcriptional activity regulates the efficiency of our secretion-expression system.

Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases

We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extracellular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride. By using TEM beta-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in beta-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of beta-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor α (MFα). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 μg/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg 25Lys 26↓Lys 27), resembles that recognized by the KEX2 gene product on which the MFα expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.

Efficient Extracellular Expression of a Foreign Protein in Streptomyces Using Secretory Protease Inhibitor (SSI) Gene Fusions

Using the gene for a secreted protease inhibitor, Streptomyces subtilisin inhibitor (SSI), we have established a secretory expression system for the products of foreign genes in Streptomyces. A sex pheromone peptide, cADl, of Enterococcus faecalis was expressed and secreted as a stable complete fusion protein with SSI in amounts comparable to the native SSI in S. lividans 66. Recombinant cADl, isolated from the fusion protein by chemical digestion with BrCN, has the same amino acid composition and sequence, retention time on reverse phase HPLC, molecular mass, and biological activity as authentic cADl. This system should be useful for the efficient expression of other foreign gene products, and as a model for heterologous gene expression in Streptomyces.

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.

High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus S-3253.

A secretory expression system for Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host, Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in S.lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain, S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from S. lividans 66 contained three additional amino acid residues in the NH2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from S. lividans 66 were found to be identical with those of authentic SSI.