Factor Xa Cleavage Site Research Articles

Structure-function analysis of muscarinic receptors and their associated G proteins

Each member of the muscarinic receptor family (M1-M5) can interact only with a limited subset of the many structurally closely related heterotrimeric G proteins expressed within a cell. To understand how this selectivity is achieved at a molecular level, we have used the G i/o-coupled M2 and the G q/11-coupled M3 muscarinic receptors as model systems. We developed a genetic strategy involving the coexpression of wild type or mutant muscarinic receptors with hybrid or mutant G protein α subunits to identify specific, functionally relevant receptor/G protein contact sites. This approach led to the identification of N- and C-terminal amino acids on α q and α i that are critical for maintaining proper receptor/G protein coupling. Moreover, several receptor sites were identified that are likely to be contacted by these functionally critical Gα residues. To gain deeper insight into muscarinic receptor structure, we recently developed a cysteine disulfide crosslinking strategy, using the M3 muscarinic receptor as a model system. Among other structural modifications, this approach involves the removal of most native cysteine residues by site-directed mutagenesis, the insertion of three factor Xa cleavage sites into the third intracellular loop, and systematic ‘reintroduction’ of pairs of cysteine residues. Following treatment of receptor-containing membrane preparations with factor Xa and oxidizing agents, disulfide cross-linked products can be identified by immunoprecipitation and immunoblotting studies. This approach should greatly advance our knowledge of the molecular architecture of muscarinic and other G protein-coupled receptors.

Expression of recombinant psoriasis-associated fatty acid binding protein in Escherichia coli: gel electrophoretic characterization, analysis of binding properties and comparison with human serum albumin.

The psoriasis-associated fatty acid binding protein (PA-FABP, also known as FABP5) is a novel keratinocyte protein that is highly up-regulated in psoriatic plaques (P. Madsen, H. H. Rasmussen, H. Leffers, B. Honoré and J. E. Celis, J. Invest. Dermatol. 1992, 99, 299-305). Here we have expressed PA-FABP in Escherichia coli as a fusion protein containing an NH2-terminal hexa-His tag followed by a factor Xa cleavage site. The recombinant protein was expressed at a level of about 30% of the soluble proteins and was purified to homogeneity using a simple two-step protocol consisting of affinity chromatography on Ni2+-nitrilotriacetic acid agarose followed by gel filtration. The recombinant protein was then digested with factor Xa and characterized by two-dimensional gel electrophoresis. The ability of PA-FABP to bind saturated fatty acids ranging from 6 to 16 carbons was determined directly by dialysis and compared to human serum albumin (HSA). The results showed that PA-FABP binds multiple molecules of the fatty acids hexanoate (C6:0), octanoate (C8:0), decanoate (C10:0) and laurate (C12:0), all with a K1 of about 10(4) M(-l), and myristate (C14:0) with a K1 of 4.4 X 10(5) M(-l). Palmitate (C16:0) also bound strongly with multiple molecules. Due to the very low solubility of palmitate its affinity to PA-FABP was measured relatively to HSA and found to be 8.1 times lower. At ligand/protein ratios below 1, all fatty acids bound to PA-FABP with about one to three orders of magnitude lower affinity than to HSA. The difference in the fatty acid binding properties of the two proteins may reflect differences in their three-dimensional structures, which in the case of PA-FABP consists mainly of beta-sheets while HSA contains predominantly alpha-helices.

An improved method to obtain a single recombinant vasoactive intestinal peptide (VIP) analog

The vasoactive intestinal peptide (VIP) is an ubiquitous peptide of great potential for applications. Development of new bioactive VIP analogs using production in recombinant E coli has been carried out in our laboratory. This work presents a new multimeric fusion protein expressing several VIP units separated by factor Xa cleavage site linkers. The steps leading from the affinity purification of the fusion protein and its processing by the factor Xa to the full characterization of the new bioactive improved VIP analog are also described.

Factor Xa Cleavage of Tissue Factor Pathway Inhibitor Is Associated with Loss of Anticoagulant Activity

Tissue factor:factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa:TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa:TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa:TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Argl99-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa:TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa:TFPI towards TF:factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF:factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.

Proper Spacing between Heptad Repeat B and the Transmembrane Domain Boundary of the Paramyxovirus SV5 F Protein Is Critical for Biological Activity

The paramyxovirus, simian virus 5, fusion (F) protein contains seven amino acids between heptad repeat B (a domain required for a biologically active fusion protein) and the presumptive boundary of the transmembrane (TM) domain. The role of the seven membrane proximal residues in stability and fusion promotion was examined by construction of a series of insertion, substitution, and deletion mutants, as manipulation of this region to enable proteolytic cleavage would facilitate production of a soluble F protein. The majority of the mutant F proteins both oligomerized and had kinetics of intracellular transport similar to those of wild-type (wt) F protein. All mutant F proteins were expressed at the cell surface at or near the same level as the wt F protein. However, by using both a qualitative lipid mixing assay and a quantitative content mixing assay for membrane fusion, it was found that mutant F proteins containing insertions in the region between heptad repeat B and the TM domain were unable to induce fusion, whereas the mutant F proteins containing substitutions in this region, together with three of the four mutants with deletions in this region, could induce fusion. Four of the F protein mutants contained a Factor Xa cleavage site, IEGR; however, Factor Xa treatment of cell surfaces released either none or only very small amounts (<1% of total protein) of the soluble heterodimer F1+ F2. As an alternative method of generating soluble F protein, a glycosyl phosphatidylinositol (GPI) anchor was added to the F protein at three membrane-proximal positions. The highest level of surface expression was observed when the final molecule did not contain a significant insertion of amino acids into the membrane proximal region. Two F-GPI mutants reached the surface at approximately 20% of the levels seen with the wt F protein, and approximately 25% of the cell surface population of these mutants could be cleaved with phosphatidylinositol phospholipase C (PI-PLC) to yield soluble F protein. However, all the F-GPI mutants oligomerized aberrantly and failed to promote fusion. Taken together, these data indicate that the spacing of the region immediately adjacent to the presumptive boundary of the TM domain is extremely important for the fusogenic activity of the SV5 F protein.

N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat.

N-terminal domains of the Wilson's and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized. Both N-MNK and N-WND bind copper specifically as indicated by the results of metal-chelate chromatography, direct copper-binding measurements, and chemical modification of Cys residues in the presence of different heavy metals. When E. coli cells are grown in the presence of copper, N-MNK and N-WND bind copper in vivo with stoichiometry of 5-6 nmol of copper/nmol of protein. Copper released from the copper-N-MNK and copper-N-WND complexes reacts with the Cu(I)-selective chelator bicinchoninic acid in the absence of reducing agents. This suggests that in proteins, it is bound in reduced Cu(I) form, in agreement with the spectroscopic properties of the copper-bound domains. Copper bound to the domains in vivo or in vitro specifically protects the N-MNK and N-WND against labeling with the cysteine-directed probe; this indicates that Cys residues in the repetitive motifs GMTCXXCXXXIE are involved in coordination of copper. Direct involvement of the N-terminal domains in the binding of copper suggests their important role in copper-dependent functions of human copper-transporting adenosine triphosphatases (Wilson's and Menkes disease proteins).

The localization of the phosphorylation site of BglG, the response regulator of the Escherichia coli bgl sensory system.

BglG, the response regulator of the bgl sensory system, was recently shown to be phosphorylated on a histidine residue. We report here the localization of the phosphorylation site to histidine 208. Localization of the phosphorylated histidine was carried out in two steps. We first engineered BglG derivatives with a specific protease (factor Xa) cleavage site that allowed asymmetric splitting of each prephosphorylated protein to well defined peptides, of which only one was labeled by radioactive phosphate. This allowed the localization of the phosphorylation site to the last 111 residues. Subsequently, we identified the phosphorylated histidine by mutating each of the three histidines located in this region to an arginine and following the ability of the resulting mutants to be in vivo regulated and in vitro phosphorylated by BglF, the bgl system sensor. Histidine 208 was the only histidine which failed both tests. The use of simple techniques to map the phosphorylation site should make this protocol applicable for the localization of phosphorylation sites in other proteins. The bgl system represents a new family of sensory systems. Thus, the mapping reported here is an important step toward the definition of the functional domains involved in the transduction of a signal by the components that constitute systems of this novel family.

Recombinant expression and isolation of human L-arginine:glycine amidinotransferase and identification of its active-site cysteine residue.

Creatine and its phosphorylated form play a central role in the energy metabolism of muscle and nerve tissues. l-Arginine:glycine amidinotransferase (AT) catalyses the committed step in the formation of creatine. The mitochondrial and cytosolic forms of the enzyme are believed to derive from the same gene by alternative splicing. We have expressed recombinant human AT in Escherichia coli with two different N-termini, resembling the longest two forms of the enzyme that we had isolated recently from porcine kidney mitochondria as a mixture. The enzymes were expressed with N-terminal histidine tags followed by factor Xa-cleavage sites. We established a new method for the removal of N-terminal fusion peptides by means of an immobilized snake venom prothrombin activator. We identified cysteine-407 as the active-site residue of AT by radioactive labelling and isolation of labelled peptides, and by site-directed mutagenesis of the protein.

Drosophila ribosomal protein PO contains apurinic/apyrimidinic endonuclease activity.

Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to generate polyclonal antibodies for Western analysis. Biochemical tests were originally performed to see if overexpressed PO contained DNase activity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3. The overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3. As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glutathione S-transferase (GST) fusions containing a Factor Xa cleavage site between GST and PO were constructed, overexpressed in an E.coli strain defective for the major 5'-acting AP endonucleases and the fusions purified using glutathione-agarose affinity column chromatography. Isolated fractions containing purified GST-PO fusion proteins were subsequently found to have authentic AP endonuclease activity. Moreover, glutathione-agarose was able to deplete AP endonuclease activity from GST-PO fusion protein preparations, whereas the resin was ineffective in lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage. These results suggested that PO was a multifunctional protein with possible roles in DNA repair beyond its known participation in protein translation. In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonucleases from sensitivity to an alkylating agent. We furthermore show that GST-PO can be located in both the nucleus and ribosomes. Its nuclear location can be further traced to the nuclear matrix, thus placing PO in a subcellular location where it could act as a DNA repair protein. Other roles beyond DNA repair seem possible, however, since GST-PO also exhibited significant nuclease activity for both single- and double-stranded DNA.

Hamster UDP-N-Acetylglucosamine:Dolichol-P N-Acetylglucosamine-1-P Transferase Has Multiple Transmembrane Spans and a Critical Cytosolic Loop

UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT) is an endoplasmic reticulum (ER) enzyme responsible for synthesis of GlcNAc-P-P-dolichol, the committed step of dolichol-P-P-oligosaccharide synthesis. The sequence of hamster GPT predicted multiple transmembrane segments (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250-14255). GPT has also been predicted to act on the cytosolic face of the ER membrane, based on topological studies of its substrates and products. In this report we test these predictions by: (i) immunofluorescence microscopy with antibodies specific for native GPT sequences or epitope tags inserted into GPT, after selective permeabilization of the plasma membrane with digitonin; (ii) insertion of Factor Xa cleavage sites; (iii) in vitro translation of GPT; and (iv) site-directed mutagenesis. The loops between the 1st and 2nd and between the 9th and 10th predicted transmembrane spans of GPT were found to be cytosolic. In contrast, the loop between the 6th and 7th transmembrane spans, as well as the carboxyl terminus, were lumenal. Thus, hamster GPT must cross the ER membrane at least three times, consistent with previous computer-assisted predictions. There was no apparent N-glycosylation or signal sequence cleavage detected by in vitro translation. The cytosolic loop between the 9th and 10th transmembrane spans is the largest hydrophilic segment in GPT and, as judged by site-directed mutagenesis, has a number of conserved residues essential for activity. Hence, these results directly support the hypothesis that dolichol-P-P-oligosaccharide assembly is initiated in the cytosol and that a downstream intermediate must translocate to the lumenal face of the ER membrane.

Genetic Analysis and Functional Characterization of Prothrombins Corpus Christi (Arg382-Cys), Dhahran (Arg271-His), and Hypoprothrombinemia

The structural abnormalities and functional characteristics of dysfunctional prothrombin variants in two new kindreds have been determined. Prothrombin Corpus Christi (family 1) was purified and found to have markedly reduced fibrinogen clotting activity, yet normal amidolytic and near-normal platelet aggregating activity. A transition (C to T) at nucleotide position 8885, present in the heterozygous form in affected family members, resulted in the substitution of Cys for Arg 382. This substitution results in the loss of a positive charge within the fibrinogen-binding exosite of thrombin, thus accounting for the observed functional defect. A heterozygous C to T transition was also present at position 19994 in other family members with a hypoprothrombinemic phenotype. This mutation results in the replacement of Gln 541 (CAA) by a premature stop codon (TAA). Prothrombin Dhahran (family 2) was found to have markedly reduced fibrinogen clotting activity, but normal amidolytic activity. Affected family members were found to have a G to A transition at nucleotide position 7312 resulting in the substitution of His for Arg 271. This substitution results in the abolition of a factor Xa cleavage site, yielding meizothrombin rather than thrombin, on activation of prothrombin Dhahran by factor Xa. All but one of the above mutations occur at CpG dinucleotides, thus further supporting the observation of a high incidence of CpG transitions in hereditary dysprothrombinemia. The significant bleeding tendencies of individuals homozygous for prothrombin Dhahran (prothrombin clotting activity 5% to 7%) contrast sharply with the absence of significant chronic bleeding in the proband expressing prothrombin Corpus Christi (prothrombin clotting activity 2%). Our findings underscore the capacity of thrombin to contribute to clinical hemostasis by mechanisms other than its fibrinogen clotting activity.

Introducing StuI sites improves vectors for the expression of fusion proteins with factor Xa cleavage sites

The Arg-encoding triplet (AGG) in the recognition sequence Ile-Glu-Gly-Arg for factor Xa can be used to generate a StuI restriction site (AGGCCT) which greatly facilitates the construction of DNA fragments encoding fusion proteins. Following proteolytic cleavage with factor Xa, a protein with the desired N terminus can be obtained

Efficient Synthesis of a 72-kDa Mitochondrial Polypeptide Using the Yeast Ty Expression System

Using the Ty system from yeast we report the efficient expression of a heterologous eukaryotic gene encoding a 72 kDa mitochondrial polypeptide. The pFM2IIBglII expression vector was initially modified for this purpose by inserting the factor Xaprotease cleavage site. TheTyAgene, which encodes the structural component of the yeast virus-like particles (VLPs), and the eukaryoticyst1 gene, encoding a 72 kDa mitochondrial tyrosyl-tRNA synthetase from the filamentous fungusPodospora anserina, were subsequently fused to the factor Xacleavage site. The resulting chimeric gene, in which the two polypeptide coding sequences are separated by the factor Xacleavage site, was expressed in yeast. High yield expression of this foreign protein, which was isolated from yeast transformants as hybrid TyVLPs, was verified after factor Xatreatment by SDS polyacrylamide gel electrophoresis and antibody detection. The strategy presented here should be useful for expressing a wide variety of eukaryotic genes.

Expression, purification, and use as an antigen of recombinant sugarcane mosaic virus coat protein.

A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.

Thrombin interaction with a recombinant N-terminal extracellular domain of the thrombin receptor in an acellular system.

The cDNA of the human endothelial cell thrombin receptor has been cloned and a chimeric fusion protein consisting of glutathione-S-transferase (GST) and the portion 25-97 corresponding to the N-terminal first extracellular domain of the thrombin receptor (TRE) has been expressed in Escherichia coli. Introduction of a factor Xa cleavage site in the fusion protein allowed purification of TRE after removal from the GST carrier protein. Purified GST-TRE or TRE have been tested in solution for their ability to interact with thrombin. alpha-Thrombin cleaved the fusion protein at position Arg-41-Ser-42 of TRE in a time- and concentration-dependent manner and GST-TRE competed with the tripeptidic substrate S-2238 for hydrolysis by thrombin (Ki = 0.5 microM). gamma-Thrombin that lacks the anion-binding exosite was 100-fold less potent than alpha-thrombin at cleaving GST-TRE. TRE competed with polymerizing fibrin monomers for binding to thrombin (Ki = 7.5 microM). The cleavage of GST-TRE by alpha-thrombin was inhibited by several alpha-thrombin exosite ligands such as the C-terminal peptide of hirudin, thrombomodulin and fibrin(ogen) fragment E. In contrast, platelet glycocalicin did not inhibit GST-TRE cleavage. In conclusion, the use of purified soluble GST-TRE allowed us to derive an affinity constant for thrombin interaction with the N-terminal domain of the receptor and to confirm the location of the cleavage site at Arg41-Ser-42 of the receptor. The importance of the thrombin anion-binding exosite for thrombin receptor recognition is highlighted by the low reactivity of gamma-thrombin for GST-TRE and by competition experiments, which in addition indicate that binding sites for fibrin(ogen), thrombomodulin and GST-TRE are overlapping. In contrast, binding of thrombin to GST-TRE and glycocalicin are not mutually exclusive, indicating that glycocalicin and TRE interact with discrete subsites within the large groove that constitutes the anion-binding exosite.

Recombinant Porcine Collagenase: Purification and Autolysis

Collagenase is a member of the matrix metalloproteinase family whose members are all capable of degrading extracellular matrix components. The mature form of porcine collagenase has been expressed in Escherichia coli using the pAX5 expression vector. The fusion protein consists of β-galactosidase at the N-terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to an active form of collagenase. Recombinant collagenase was biologically active in the form of a fusion protein; this was cleaved with factor Xa to yield collagenase with the authentic N terminus (phenylalanine) found in vivo and purified in a single step on a peptide hydroxamic acid affinity column. On purification the recombinant porcine collagenase undergoes autolysis at a number of different bonds in the region connecting the active site domain with the C-terminal hemopexin-like domain. This may represent a loop region of poor secondary structure, making it susceptible to relatively nonspecific cleavage, The N-terminal fragment retains a reduced level of collagenolytic activity, along with that against casein and gelatin.

Prothrombin Padua I: incomplete activation due to an amino acid substitution at a factor Xa cleavage site

An individual and an affected brother previously identified as having the variant prothrombin Padua I were studied in order to identify underlying genetic defects. A heterozygous mutation in the prothrombin gene exon 8 was identified as substitution of A for G at nucleotide position 7,312 (Arg271 (CGT) to His (CAT)). An abolished RsaI restriction site was used to confirm heterozygosity for the defect. Lack of the requisite cleavage of the His271-Thr272 bond in prothrombin Padua I could prevent release of fragment 2 and block the conversion of the intermediate meizothrombin des fragment 1 to alpha-thrombin, providing an explanation of reduced potential for clotting activity and for the observed mild bleeding tendency.

Expression and export in Escherichia coli of fusion proteins containing carboxy-terminally located honeybee prepromelittin.

The aim of this work was to express a eukaryotic pre-protein in Escherichia coli so that it could be obtained intact, without cleavage, by bacterial leader peptidase. To this end, cDNA coding for honeybee prepromelittin was ligated to the 3' end of genes coding for truncated forms of either Protein A or beta-galactosidase (beta-Gal) under the control of inducible promoters, with an oligonucleotide coding for the Factor Xa cleavage site at the junction between the two proteins. The Protein A fusion was expressed in good yield, and about 80% of it formed inclusion bodies. The prepromelittin section of the Protein A fusion caused some export of the intact fusion protein to the growth medium. The prepromelittin beta-Gal fusion was expressed in low yield and became associated with the E. coli cytoplasmic membrane. Its expression was toxic to E. coli. Thus, the synthesis of a full-length eukaryotic pre-protein in E. coli is best achieved when the fusion protein forms inclusion bodies.

Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis

For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R−1 and I+1. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A−4-E-P-R−1- to -A−4-E-P-Q−1- by site-directed mutagenesis.

High Yield of Active STb Enterotoxin from a Fusion Protein (MBP-STb) Expressed in Escherichia coli

Maltose binding protein (MBP) fused to STb, a heat-stable enterotoxin of Eseherichia coli, was secreted into the periplasm. A factor Xa cleavage site is present between MBP and STb allowing MBP to be cleaved from STb. The gene fusion is under the control of the strong and inducible P tac, promoter. Three hours after induction with IPTG, cells were harvested. Following osmotic shock treatment of the cells, the MBP-STb fusion protein was released and affinity-purified using an amylose resin. The fusion protein purified in this way was biologically active in ligated intestinal segments of rats. Digestion of MBP-STb with factor Xa released native STb which was purified to homogeneity by reverse-phase chromatography using a PepRPC column. The toxin was eluted at approximately 38% acetonitrile. The 5000-Da toxin was shown to be pure by SDS- PAGE and immunoblotting. The recovered enterotoxin was active in the rat loop assay. Amino acid sequence analysis showed that the first eight residues were identical to those of native STb, confirming the identity of STb. The ultraviolet absorption spectra of purified STb revealed low absorption at 254 and 280 nm compared to 210-230 nm. Isoelectric focusing under nondenaturing conditions indicated a p I of 9.6. Typically, 8 liters of bacterial culture resulted in 2.2 mg of pureSTb. This genetic construction provides a readily obtainable source of biologically active STb toxin.