Recognition of cello-oligosaccharides by a family 17 carbohydrate-binding module: an X-ray crystallographic, thermodynamic and mutagenic study.

The crystal structure of the Clostridium cellulovorans carbohydrate-binding module (CBM) belonging to family 17 has been solved to 1.7 A resolution by multiple anomalous dispersion methods. CBM17 binds to non-crystalline cellulose and soluble beta-1,4-glucans, with a minimal binding requirement of cellotriose and optimal affinity for cellohexaose. The crystal structure of CBM17 complexed with cellotetraose solved at 2.0 A resolution revealed that binding occurs in a cleft on the surface of the molecule involving two tryptophan residues and several charged amino acids. Thermodynamic binding studies and alanine scanning mutagenesis in combination with the cellotetraose complex structure allowed the mapping of the CBM17 binding cleft. In contrast to the binding groove characteristic of family 4 CBMs, family 17 CBMs appear to have a very shallow binding cleft that may be more accessible to cellulose chains in non-crystalline cellulose than the deeper binding clefts of family 4 CBMs. The structural differences in these two modules may reflect non-overlapping binding niches on cellulose surfaces.

Crystallization and preliminary X-ray diffraction analysis of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the bacterium Cellulomonas fimi.

Single crystals of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the cellulolytic bacterium Cellulomonas fimi, have been grown in the presence of polyethylene glycol 4000 using the vapour diffusion technique. The crystals, which diffract to better than 2.0 A resolution, belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have cell constants: a = b = 88.21 A, c = 81.10 A; alpha = beta = gamma = 90 degrees.

Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification.

The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the beta-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604-607], each of the exposed tryptophans interacts reversibly with cellulose.

The expression of Cellulomonas fimi cellulase genes in Brevibacterium lactofermentum.

The exoglucanase gene (cex) and the endoglucanase A gene (cenA) from Cellulomonas fimi were subcloned into the Escherichia coli/Brevibacterium lactofermentum shuttle vector pBK10. Both genes were expressed to five to ten times higher levels in B. lactofermentum than in E. coli, probably because these genes were expressed from C. fimi promoters. In B. lactofermentum virtually all of the enzyme activities were in the culture supernatant. This system will facilitate analysis of the expression of the C. fimi genes in and secretion of their products from a Gram-positive bacterium.

Structure of the gene encoding the exoglucanase of Cellulomonas fimi.

In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.

Secretion of Cellulomonas fimi exoglucanase by Escherichia coli.

A leader sequence of 41 amino acids (aa) has been proposed as the signal sequence for the exoglucanase (Exg) from Cellulomonas fimi. The ability of this 41-aa peptide to function as a leader sequence has been shown here by gene fusion experiments in Escherichia coli. A hybrid leader sequence containing C-terminal 37 aa of the leader peptide and N-terminal 6 aa of beta-galactosidase (beta Gal) directed export of the Exg into the periplasm of E. coli. In contrast, hybrid beta Gal-Exg proteins in which the leader sequence is not present are retained in the cytoplasm.

Endoglucanase CasA from alkalophilic Streptomyces strain KSM-9 is a typical member of family B of beta-1,4-glucanases.

CasA is an endo-beta-1,4-glucanase from Streptomyces KSM-9 belonging to family B of beta-1,4-glucanases. A previous analysis of a portion of the corresponding gene (casA) revealed sequencing errors in a region encoding part of the catalytic site. Additional errors in the original sequence were suspected, based on sequence comparison of the C terminus of CasA with other members of its family. Re-sequencing of the remainder of the casA coding region showed that CasA is a typical member of family B.

The pTugA and pTugAS vectors for high-level expression of cloned genes in Escherichia coli.

Plasmids pTugA and pTugAS, designed for expression of cloned genes in Escherichia coli, possess the features of high-level inducible transcription, enhanced RNA translation, portability, high copy number, stability and versatility. In addition, pTugAS can be used to produce fusion proteins comprising a target protein and a cellulose-binding domain. Such fusion proteins can be purified in a single step by affinity chromatography on cellulose. Expression of two model gene fusions using the pTug plasmids resulted in yields of 500 mg of intracellular and 250 mg of extracellular recombinant protein per liter.

Molecular cloning of a Cellulomonas fimi cellulose gene in Escherichia coli.

A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant DNA plasmids carrying a cellulase gene. The assay was used to identify a recombinant DNA plasmid carrying at least one cellulase gene from Cellulomonas fimi. The enzyme present in extracts of E. coli carrying the plasmid was active in catalysing the hydrolysis of carboxymethylcellulose as indicated by the production of reducing sugars.

Streptomyces lividans glycosylates an exoglucanase (Cex) from Cellulomonas fimi.

Exoglucanase Cex from Cellulomonas fimi is a glycoprotein [Langsford et al., J. Gen. Microbiol. 130 (1984) 1367-1376]. Cex produced by Streptomyces lividans from the cloned cex gene is also glycosylated. The extent and nature of glycosylation are similar for Cex from both organisms. The glycosylation affords protection against proteolysis for the enzymes from both organisms when they are bound to cellulose, but not in solution. The ability to glycosylate cloned gene products enhances the utility of Streptomyces as a host for the production of heterologous polypeptides.

Characterization and structure of an endoglucanase gene cenA of Cellulomonas fimi.

Two BamHI fragments (0.8 and 5.2 kb) of Cellulomonas fimi containing an endoglucanase (Eng) gene (cenA) were individually cloned into the BamHI site of pBR322; they expressed carboxymethylcellulase activity in Escherichia coli. The nucleotide (nt) sequence of the cenA gene was determined by sequencing overlapping deletions. The cenA gene is 1350 bp long encoding a polypeptide of 449 amino acids (aa) and stop codon. The 0.8-kb BamHI component encodes the first 76 aa, whereas the 5.2-kb BamHI component encodes the rest of the Eng. The Eng lacking the N-terminal 76 aa retains its activity and antigenicity, and it forms an active fusion protein with the N-terminal portion of the TcR determinant. The C-terminal region of the Eng is crucial for activity and a deletion of as little as 12 aa from that end results in the loss of all Eng activity. The N-terminal 31 aa of the Eng constitute a leader peptide which appears to be functional in exporting the enzyme to the periplasm in E. coli.

A bifunctional exoglucanase-endoglucanase fusion protein.

A fusion was constructed between the cex gene of Cellulomonas fimi, which encodes an exoglucanase, and the cenA gene of the same organism, which encodes an endoglucanase. The cex-cenA fusion was expressed in Escherichia coli to give a fusion protein with both exoglucanase and endoglucanase activities. The fusion protein, unlike the cex and the cenA gene products from E. coli, did not bind to microcrystalline cellulose, presumably because it lacked an intact substrate-binding region. The fusion protein was exported to the periplasm in E. coli.

Fusion to an endoglucanase allows alkaline phosphatase to bind to cellulose.

Endoglucanase CenA of Cellulomonas fimi comprises an N-terminal cellulose-binding domain and a C-terminal catalytic domain joined together by a sequence of 23 proline and threonine residues (the Pro-Thr box). The domains function independently when separated by proteolysis. TnphoA has been used to generate cenA'-'phoA fusions. CenA'-'PhoA fusion polypeptides which contain the entire cellulose-binding domain of CenA bind to cellulose, allowing their purification from periplasmic extracts in a single, facile step. This result has implications for purification or immobilisation of chimeric proteins on a cheap cellulose matrix.

Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains.

Glycosylated cellulases from Cellulomonas fimi were compared with their non-glycosylated counterparts synthesized in Escherichia coli from recombinant DNA. Glycosylation of the enzymes does not significantly affect their kinetic properties, or their stabilities towards heat and pH. However, the glycosylated enzymes are protected from attack by a C. fimi protease when bound to cellulose, while the non-glycosylated enzymes yield active, truncated products with greatly reduced affinity for cellulose.

Quantitation of helper factor activity. II. Inhibition of antibody binding.

A sensitive direct assay has been developed to quantitate antigen specific helper factors (ThF). The assay depends on the ability of ThF to block the binding of free antibody to immobilized antigen. It has been used to follow the course of purification of ThF produced in short-term cultures of in vitro primed helper cells and purified by affinity chromatography on antibody and antigen immunoabsorbents. The identity of the ThF was confirmed by assaying its biological activity and by its reactivity with anti-Ia antibody and with a monoclonal anti-ThF.

Quantitation of helper factor activity. I. Utilization of the ELISA method to measure helper activity in the secondary antibody response.

We describe here a simple culture system for measuring the effects of regulatory cells or factors on the secondary antibody response which uses the enzyme-linked immunosorbent assay (ELISA) to detect antibody production. This technique offers several advantages over methods which measure hemolytic plaque-forming cells. Helper factors purified from the supernatant of in vitro primed helper cells were shown to specifically enhance antibody production from primed spleen cells by from 2- to 4-fold in this system.

Mannanase Man26A from Cellulomonas fimi has a mannan-binding module.

A modular mannanase (Man26A) from the bacterium Cellulomonas fimi contains a mannan-binding module (Man26Abm) that binds to soluble but not to insoluble mannans. Man26Abm does not bind to cellulose, chitin or xylan. The K(d) for binding of Man26Abm to locust bean gum (LBG) is approximately 0.2 microM. Man26A is the first mannanase reported to contain a mannan-binding module.

Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae.

The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar. The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine. The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine. IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA. When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N. gonorrhoeae proteases. It is cleaved within the hinge sequence at the same sites as IgA1.

Rapid immunoassay technique for process monitoring of animal cell fermentations.

A calibration and quality control technique suited to process monitoring with immunoassay is demonstrated. The particle concentration fluorescence immunoassay (PC-FIA) is shown to provide a sensitive and rapid method for the quantification of specific biomolecules in cell cultures. Smoothing of linear calibration parameters is performed by forming weighted averages of standard points as the run progresses. These estimates are then used to determine slope and intercept values for improved calibration. The nonuniformity of the fluorescent signal variance is also considered, and a weight model is developed to describe the relationship between signal fluorescence and signal variance for weighted linear curve fitting. Pooling calibration results over the process run improves overall assay performance as determined by using standard control chart analysis. This method is suitable for semicontinuous monitoring of animal cell fermentations and has been used here to measure cell-associated and culture supernatant concentrations of monoclonal antibody (Ab) from hybridoma cells. The cell-associated Ab concentration correlates with cell-specific production rate. Assay times on the order of 10 min for supernatant and 25-30 min for cell-associated Ab concentrations can be achieved, making this procedure suitable for process monitoring and control. Under these conditions the assay has a detection limit of approximately 10 ng/mL, providing a sensitive and specific method for the quantification of cell culture constituents.

Processing of fusion proteins with immobilized factor Xa.

Factor Xa, with a cellulose-binding domain (CBD) fused to the C-terminus of the heavy chain (FXa-CBD), is active in solution and when immobilized on cellulose. A second derivative of factor Xa in which a hexahistidine tail is fused to the C-terminus of the heavy chain (FXa-H6) also retains activity when immobilized, in this case on Ni(2+)-NTA agarose. The stabilities and activities of of FXa-CBD and FXa-H6 immobilized on cellulose and Ni(2+)-NTA agarose, respectively, are similar. Immobilized factor Xa derivatives can be used to remove affinity tags from appropriate fusion proteins without contaminating the desired product with factor Xa.