Amylose Affinity Chromatography Research Articles

Development of a method for expression and purification of the regulatory C2-like domain of human 5-lipoxygenase

5-Lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is built of a catalytic C-terminal domain and a regulatory N-terminal C2-like domain. The C2-like domain is the target of many regulatory factors or proteins including Ca2+, phospholipids, glycerides, coactosin-like protein and presumably other components that modulate the catalytic activity of 5-LO by acting at this domain, but the detailed underlying molecular mechanisms of these interactions are still unclear. In order to obtain the 5-LO C2-like domain as purified protein in good yields for further mechanistic studies and structure elucidation, a novel expression and purification approach has been applied. A plasmid was constructed expressing a fusion protein of maltose-binding protein (MBP) and the regulatory C2-like domain of 5-LO (AS 1–128), separated by a tobacco etch virus (TEV) protease-cleavage site. The fusion protein MBP-5LO1-128 could be essentially expressed as a soluble protein in Escherichia coli and was efficiently purified by amylose affinity chromatography. By means of this procedure, approximately 80mg purified fusion protein out of 1L E. coli culture were obtained. Digestion with TEV protease yielded the C2-like domain that was further purified using hydrophobic interaction chromatography. Alternatively, the uncleaved fusion protein MBP-5LO1-128 may be suitable to immobilize the C2-like domain on an amylose resin for co-factor interaction studies. Together, we present a convenient expression and purification strategy of the 5-LO C2-like domain that opens many possibilities for structural determination and mechanistic studies, aiming to reveal the precise role and function of this regulatory domain.

Biochemical Studies of Klebsiella pneumoniae NifL Reduction Using Reconstituted Partial Anaerobic Respiratory Chains of Wolinella succinogenes

In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK(6)) or 8-methylmenaquinone (MMK(6)) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK(6) from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.

Refolding, purification and characterization of replication-initiator protein from soybean-infecting geminivirus

The replication-initiator protein (Rep) from a soybean-infecting geminivirus was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). In spite of the presence of the highly soluble MBP as the fusion partner, the overexpressed MBP-Rep fusion protein formed insoluble inclusion bodies. The protein was solubilized from the inclusion bodies and refolded. The refolded MBP-Rep protein was purified using ion exchange and amylose affinity chromatography. The activity of the purified MBP-Rep was assessed using an in vitro cleavage assay. Soluble and stable MBP-Rep protein was obtained in high abundance, providing the feasibility of large-scale production of active Rep protein for functional characterization and X-ray crystallographic structure determination.

Fusion expression ofHelicobacter pylorineutrophil-activating protein inE.coli

To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori (H pylori) neutrophil-activating protein (NAP) and E. coli maltose-binding protein (MBP) and to evaluate its immunoreactivity and immunogenicity. Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis. Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment, Western blotting with human H pylori anti-sera. E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG. The molecular weight of rBMP-NAP was about 57 kD, accounting for 37.55% of the total protein in the sonicated supernatant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture. The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself. Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.

Genomic cloning, expression and recombinant protein purification of α and β subunits of the allophycocyanin gene (apc) from the cyanobacterium Anacystis nidulans UTEX 625

A genomic fragment encoding alpha(APC) and beta(APC) (i.e., alpha and beta units of the allophycocyanin, APC) from Anacystis nidulans UTEX 625 was cloned and sequenced. This fragment, containing a non-coding sequence of 56 nucleotides in between, was then subcloned into the expression vector pMal-c2 downstream from and in frame with the malE gene of E. coli encoding MBP ( maltose binding protein). The fusion protein was purified by amylose affinity chromatography and cleaved by coagulation factor Xa. alpha(APC) and beta(APC) were then separated from MBP and MBP fusion proteins, respectively, and concentrated by membrane centrifugation. The study provides a method to produce recombinant allophycocyanin subunits for biomedical and biotechnological applications.

Soluble Expression and Affinity Purification of Functional Domain of Human Acetylcholine Receptor ?-subunit by the Modulation of Maltose Binding Protein

An open reading frame of the alpha-subunit 1-205 residues (alpha205) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR alpha205 as the template and inserted into vector pMAL-c2X. The constructed pMAR alpha205 was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR alpha205 protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR alpha205 could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR alpha205 and AChR alpha205 were similar to that of AChR alpha-subunit from Torpedo.

Cloning and expression of human colon mast cell carboxypeptidase.

To clone and express the human colon mast cell carboxypeptidase (MC-CP) gene. Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR). The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to construct prokaryotic expression vector pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP. The recombinant fusion protein expressed in E. coli was induced with IPTG and purified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography. The recombinant hMC-CP expressed in Pichia pastoris (P. pastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid sequencing and enzyme assay. The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA. The recombinant hMC-CP protein expressed in E. coli was purified with amylose affinity chromatography and heparin agarose chromatography. SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E. coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5). The N-terminal amino acid sequence confirmed further the product was hMC-CP. E. coli generated hMC-CP showed a very low level of enzymatic activity, but P. pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine. The cDNA encoding human colon mast cell carboxypeptidase can be successfully cloned and expressed in E. coli and P. pastoris, which will contribute greatly to the functional study on hMC-CP.

Phosphorylation of the Yeast Phospholipid Synthesis Regulatory Protein Opi1p by Protein Kinase A

The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-containing genes involved in phospholipid synthesis in the yeast Saccharomyces cerevisiae. The phosphorylation of Opi1p by protein kinase A (cAMP-dependent protein kinase) was examined in this work. Using a maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase A activity was time- and dose-dependent and dependent on the concentrations of Opi1p and ATP. Protein kinase A phosphorylated Opi1p on multiple serine residues. The synthetic peptides SCRQKSQPSE and SQVRESLLNL containing the protein kinase A motif for Ser31 and Ser251, respectively, within Opi1p were substrates for protein kinase A. Phosphorylation of S31A and S251A mutant maltose-binding protein-Opi1p fusion proteins by protein kinase A was reduced when compared with the wild type protein, and phosphopeptides present in wild type Opi1p were absent from the S31A and S251A mutant proteins. In vivo labeling experiments showed that the extent of phosphorylation of the S31A and S251A mutant proteins was reduced when compared with the wild type protein. The physiological consequence of the phosphorylation of Opi1p at Ser31 and Ser251 was examined by measuring the effects of the S31A and S251A mutations on the expression of the UASINO-containing gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacZ reporter gene in opi1Delta mutant cells expressing the S31A and S251A mutant Opi1p proteins was elevated 42 and 35%, respectively, in the absence of inositol and 55 and 52%, respectively, in the presence of inositol when compared with cells expressing wild type Opi1p. These data supported the conclusion that phosphorylation of Opi1p at Ser31 and Ser251 mediated the stimulation of the negative regulatory function of Opi1p on the expression of the INO1 gene.

Production of extracellular domain of human tissue factor using maltose-binding protein fusion system

Making use of the physiological process of coagulation as an anti-tumor effector function may be beneficial in various coagulation-mediated diseases. Preclinical and clinical studies with novel tissue factor targeting constructs require that efficient procedures for preparing large quantities of pure truncated TF (tTF) become available. In this study, we described a simple and rapid on-column method for purifying large quantities of human tTF from Escherichia coli. The coding region of extracellular domain of tissue factor was linked to the 3 ′-end of maltose-binding protein (MBP) gene. The fusion protein was expressed as soluble form after induction by isopropylthio-β- d-galactoside (IPTG). MBP-tTF was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 21.5% of the total soluble protein in E. coli, allowing approximately 15 mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-TF monoclonal antibody and the activity was confirmed by chromogenic assay. This MBP-fusion system permits large-scale functional expression and purification of recombinant soluble proteins, providing a basis for the future study of structure and function of tTF.

Raising antibodies against OprD, an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE

OprD is an outer membrane porin of Pseudomonas aeruginosa that mediates uptake of basic amino acids, peptides as well as carbapenem antibiotics. Polyclonal antibodies were raised against the OprD porin by creating protein fusions between the Escherichia coli maltose binding protein and four OprD fragments. These were expressed in E. coli and shown to be exported to the periplasm. The fusion proteins were purified by amylose affinity chromatography and used to immunize rabbits intramuscularly. We established that MalE fusions to OprD fragments retain maltose and amylose binding activities in vivo and in vitro, confirming proper folding of the MalE domain of hybrid proteins. Furthermore, we demonstrate that this strategy can be used to obtain specific antibodies against bacterial outer membrane proteins (OMPs).

Production and purification of single chain human insulin precursors with various fusion peptides

For the production and purification of a single chain human insulin precursor four types of fusion peptides β-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)6-tagged sequence (HTS) were investigated. RecombinantE. coli harboring hybrid genes was cultivated at 37°C for 1 h, and gene induction occurred when 0.2 mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except forE. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0 mM IPTG, followed by a longer than 4 h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique

Protein purification with C-terminal fusion of maltose binding protein.

For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus. In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of the plant photoreceptor phytochrome B allows purification of the fusion protein via amylose affinity chromatography. After overexpression in yeast a 125-fold enrichment could be achieved. The spectral properties of phytochrome B were not impaired by the fusion and purification. These results demonstrate that not only the widely used N-terminal fusions of MBP but also C-terminal fusions can be employed for protein purification.

Purification and Characterization of the Periplasmic Domain of EnvZ Osmosensor inEscherichia coli

The EnvZ-OmpR histidyl-aspartyl phosphorelay system inE. coliresponds to osmolarity by differentially modulating the expression of the major outer membrane porins OmpF and OmpC. To date, the natural ligand that activates EnvZ, a transmembrane histidine kinase, has not been identified and the role of the periplasmic domain of EnvZ is unclear. We now report on the purification and characterization of the periplasmic domain of EnvZ (Lys48-Arg162) which has been expressed as a soluble protein in fusion with the maltose-binding protein. Overexpression of the fusion protein did not compete for a signal that activates EnvZ. By amylose affinity chromatography and affinity blotting, interacting proteins could not be detected. The periplasmic domain was released by factor Xa and purified to homogeneity. From circular dichroism analysis, the periplasmic domain was estimated to consist of 35% α-helices and 16% β-sheets.

The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions.

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE-PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE-PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE-PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fattyacylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS-like chaperone proteins.

Characterization of Purified Recombinant Bet v 1 with Authentic N-Terminus, Cloned in Fusion with Maltose-Binding Protein

A gene encoding the pollen major allergen Bet v 1 fromBetula verrucosa(White Birch) has been cloned and expressed inEscherichia colias a fusion with maltose-binding protein and a Factor Xa proteolytic cleavage site. A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the authentic amino terminus of Bet v 1 was generated after cleavage. Fusion protein was isolated by amylose affinity chromatography and enzymatically cleaved by incubation with Factor Xa. Recombinant Bet v 1 was isolated by gel filtration and gave rise to a single band with apparent molecular weight of 17 kDa when analyzed by SDS–polyacrylamide gel electrophoresis. N-terminal sequencing of the first 20 amino acids showed complete agreement with the deduced Bet v 1 DNA sequence. Mass spectrometry showed that recombinant Bet v 1 has a molecular mass of 17,440 ± 2 Da; 86% of the recombinant Bet v 1 amino acid sequence could be verified by digestion with Lys-C and mass spectrometric peptide mapping. The yield of purified recombinant Bet v 1 was 10 mg per literE. colicell culture. Two-dimensional gel electrophoresis of purified recombinant protein gave rise to one major protein spot and one or two minor spots focusing at slightly different pHs. The immunochemical properties of recombinant protein were indistinguishable from those of naturally occurring Bet v 1 when compared using a panel of murine monoclonal antibodies and serum IgE from birch pollen allergic patients. Furthermore, recombinant Bet v 1 elicited T-cell proliferation comparable to that of natural Bet v 1. Thus, the methods used for bacterial expression and protein purification result in relatively high yields of folded recombinant Bet v 1 with correct N-terminal sequence and molecular mass. Furthermore, the B- and T-cell epitope structures of recombinant Bet v 1 closely resemble those of the natural protein from pollen.

Expression and purification of the light chain of botulinum neurotoxin A: a single mutation abolishes its cleavage of SNAP-25 and neurotoxicity after reconstitution with the heavy chain.

Botulinum neurotoxin type A (BoNT/A) selectively and irreversibly inhibits acetylcholine release from peripheral nerve endings. While the toxin's heavy (H) chain contributes to neuronal binding and internalization, its light (L) chain is a Zn(2+)-dependent endoprotease that intracellularly cleaves synaptosomal-associated protein of M(r) = 25 kDa (SNAP-25). For research and clinical exploitation of this uniquely-acting neurotoxin, recombinant wild-type L chain was produced together with a mutant in which His227 in the Zn(2+)-binding motif was substituted by Tyr. The PCR-amplified wild-type and mutant L chain genes were cloned, fused to the gene for maltose-binding protein, and expressed at high levels in Escherichia coli. The soluble fusion proteins were purified using amylose affinity chromatography, and, after factor Xa cleavage, the free L chains were isolated. The wild-type was shown to proteolyze SNAP-25 at a rate approaching that of the native chain while the mutant was inactive. Reconstitution of the pure wild-type L chain with native homogeneous H chain yielded a disulfide-linked dichain form that inhibited neuromuscular transmission in vitro and produced the symptoms of botulism in vivo. After reconstitution with the H chain, the Tyr227 mutant L chain failed to show any neuroparalytic activity in either of these assays. This methodology allows, for the first time, routine preparation of recombinant forms of the L chain that are needed to decipher the molecular details of its interaction with substrate and, thereby, assist the design of effective inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and Characterization of a Single Recombinant Proteoglycan Tandem Repeat Domain of Link Protein That Binds Zinc and Hyaluronate

The first proteoglycan tandem repeat (PTR) of bovine link protein has been cloned in the pMAL-c vector and overexpressed in fusion with maltose-binding protein (MBP) in Escherichia coli. The fusion protein can be isolated from the soluble phase of the bacterial lysate by amylose affinity chromatography. The PTR domain can be cleaved from the MBP domain with factor Xa protease. Evidence using zinc affinity chromatography is presented which indicates that at least one of the zinc-binding sites of bovine link protein is contained within the first PTR domain. Zinc affinity chromatography was then incorporated as the final purification step of the MBP/PTR protein. Evidence for the binding of MBP/PTR to hyaluronic acid (HA) is demonstrated by coprecipitation with HA using cetylpyridinium chloride. Binding is specific since MBP/PTR does not coprecipitate with chondroitin sulfate. Binding is also demonstrated in an ELISA assay on HA-coated plates. In this assay, binding could be inhibited by the addition of HA or HA oligosaccharides.

Expression, purification, and characterization of a novel G protein, SGP, from Streptococcus mutans.

The sgp gene of Streptococcus mutans was recently detected immediately downstream from the dgk gene within the same operon. In this study, the sgp gene was subcloned into the pMAL-c2 vector and SGP (S. mutans G protein) was overexpressed in Escherichia coli as a fusion protein with the maltose-binding protein at a level of 40% of total cellular protein. One-step amylose affinity chromatography purification of this fusion protein yielded a product of approximately 95% purity. SGP was purified from this fusion protein following cleavage with protease factor Xa and DEAE-Sephacel chromatography. In nucleotide binding assays, recombinant SGP showed specific binding for GTP and GDP, but not ATP, CTP, and UTP, and also catalyzed efficient hydrolysis of GTP to GDP. Kinetic studies revealed that the SGP Km value for GTP in this reaction was approximately 5.9 microM. Mg2+ also served as a cofactor of SGP in this reaction. In vivo subcellular localization by immunogold labelling demonstrated that SGP was associated with both membrane and cytoplasmic fractions. SGP not only had structural similarities with other G proteins but also proved to have high-level intrinsic GTPase activity. Therefore, SGP appears to be a new member of the G protein superfamily and may participate in transmembrane signaling in the responses of S. mutans cells to environmental stimuli.

Production and purification of active FGF2 via recombinant fusion protein

Basic fibroblast growth factor (FGF2) is involved in both cell proliferation and differentiation processes. Heparin may interfere in the stability and biological activities of FGFs. However, it is difficult to obtain FGF preparation without traces of heparin since heparin affinity chromatographies are routinely used to prepare this growth factor. We have therefore devised a means of production of active recombinant FGF2 devoided of heparin traces. The bovine FGF2 gene was inserted into the pMAL-c prokaryotic expression vector and the recombinant protein was synthesised as a fusion product between the maltose binding protein (MBP) and FGF2. Purification of the FGF2 fusion protein was performed by an amylose affinity chromatography. Yields were similar to those obtained by using a traditional heparin affinity column purification procedure. The fusion protein (MBP-FGF2) and the cleaved-off FGF2 were tested for some of their biological properties and compared to recombinant FGF2 purified by heparin affinity chromatography. Mitogenic activity on Chinese hamster lung fibroblast (CCL39) and neurite outgrowth on pheochromocytoma culture cells (PC12) were used as biological assays. The cleaved-off FGF2 was as active as commercially available recombinant FGF2 (ED 50 at 0.16 and 0.04 nM respectively). However MBP-FGF2 was less active (ED 50 at 0.9 nM) in both tests.

Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide

A Neocallimastix patriciarum cDNA library was screened for xylanase-expressing clones, which were distinct from the previously characterized N. patriciarum xynA cDNA encoding xylanase A. A single cDNA, designated xynB, which did not exhibit homology with xynA, was isolated. Northern-blot analysis of mRNA from Avicel-grown N. patriciarum showed that xynB hybridized to a 3.4 kb mRNA species. The nucleotide sequence of xynB revealed a single open reading frame of 2580 bp coding for a protein designated xylanase B (XYLB), of M(r) 88,066. The primary structure of XYLB was comprised of a 21-residue N-terminal signal peptide, followed by a 304-amino acid sequence that exhibited substantial homology with the catalytic domains of family F xylanases. The N-terminal domain was linked to a C-terminal 70-residue sequence by a putative linker region, comprising 12 tandem repeats of a sequence containing TLPG as the core sequence, followed by an octapeptide XSKTLPGG where X can be S, K or N, which was repeated in tandem 45 times. Truncated derivatives of xynB encoding the N-terminal 338 residues directed the synthesis of a functional xylanase, confirming that the region of XYLB, which exhibited homology with family F xylanases, constitutes the catalytic domain. To investigate the catalytic properties of XYLB, the catalytic domain was fused to the Escherichia coli maltose-binding protein, and the fusion protein purified by amylose affinity chromatography. The purified enzyme hydrolysed oat, rye and wheat arabinoxylan releasing primarily xylobiose, xylotriose and some xylose. The XYLB fusion did not cleave any cellulosic substrates. The data presented in this report suggest that the multiple xylanases of N. patriciarum arose, not through the duplication of a single gene, but by the transfer of distinct xylanase-encoding DNA sequences into the anaerobic fungus. The possible origin of the xynB gene is discussed.