Mara Dekel

Expression, cross-linking, and characterization of recombinant chitin binding resilin.

Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance protein-carbohydrate composite material. We describe for the first time a comprehensive study on the mechanical, structural and biochemical function of chitin binding recombinant Drosophila melanogaster resilin. Various resilin constructs were cloned including the full length gene enabling Ni-NTA purification, as well as heat and salt precipitation for rapid and efficient purification. The binding isotherms and constants (Kd, Bmax) of resilin to chitin via its chitin binding domain were determined and displayed high affinity to chitin, implying its important role in the assembly of the resilin-chitin composite. The structural and elastic properties were investigated using Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and Atomic Force Microscopy (AFM) with peroxidase crosslinked solid resilin materials. Generally, little structural organization was found by these biophysical methods, suggesting structural order was not induced by the dityrosine crosslinks. Further, the elastomeric properties found from the full length protein compared favorably with the shorter resilin generated previously from exon 1. The unusual elastomeric behavior of this protein suggests possible utility in biomaterials applications.

Immobilization of recombinant heparinase I fused to cellulose-binding domain

Immobilization of biologically active proteins is of great importance to research and industry. Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins. We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD. Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD). The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies. The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column. The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin. C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose. CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity. By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled. The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared. The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization. Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step.

Cloning and characterization of elongation specific endo-1,4-β-glucanase (cel1) from Arabidopsis thaliana

The isolation of an elongation-specific endo-1,4-β-glucanase-cel1 from Arabidopsis thaliana was made possible by the fact that considerable homology exists between different endo-1,4-β-glucanase (EGase) genes from different plants. Degenerate primers were synthesized based on two conserved regions from the avocado and tomato cellulase amino acid sequences. The A. thaliana cel1 cDNA gene was found to encode a 54 kDa protein; sequence comparison with the avocado EGase revealed 56% identity. Northern blot analysis of cel1 suggested its developmental regulation. RNA transcripts were undetectable in fully expanded leaves as well as at the basal internode of flowering stems. However, a strong transcript signal was detected in the elongating zone of flowering stems of normal plants. The RNA transcript level of cel1 in the elongating zone of dwarf flowering stems was significantly lower than in the corresponding zone in normal plants. This suggests cel1s involvement in cell elongation in A. thaliana. Transgenic tobacco plants transformed with the putative cel1 promoter region fused to the gus reporter gene, showed a significant GUS staining both in shoot and root elongating zones. These results further substantiate the link between cel1 expression and plant cell elongation.

Growth enhancement of transgenic poplar plants by overexpression of Arabidopsis thaliana endo-1,4–β-glucanase (cel1)

Poplar (Populus tremula) plants which had been transformed with Arabidopsis thaliana cel1 cDNA and successfully over-expressed the gene, exhibited significant phenotypic alterations which included taller plants, larger leaves, increased stem diameter, wood volume index, dry weight and a higher percentage of cellulose and hemicellulose, compared to the wild-type plants. Transgenic A. thaliana plants over-expressing A. thaliana cel1 exhibited similar levels of cel1 mRNA in the elongation zone of the flowering stem and higher levels in mature leaves when compared with wild-type plants. CEL1 protein levels in the elongation zone of the flowering stem of transgenic plants were similar or slightly higher compared to that of the wild-type plants, whereas mature leaves of transgenic plants contained a higher level of CEL1. These data indicate that in elongating zone of Arabidopsis, CEL1 level is tightly regulated. In contrast to transgenic poplar over-expressing the A. thaliana cel1, no phenotypic difference was found between A. thaliana transgenic and wild-type plants.

Expression, purification and applications of staphylococcal Protein A fused to cellulose-binding domain

Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purification and diagnostics. Immobilization is required for most of these applications. Here we describe a genetic‐engineering approach to immobilizing ProtA on cellulose, by fusing it to cellulose‐binding domain (CBD) derived from the cellulose‐binding Protein A of Clostridium cellulovorans. The bifunctional fusion protein was expressed in Escherichia coli, recovered on a cellulose column and purified by elution at alkaline pH. ProtA–CBD was used to purify IgG from rabbit serum and its ability to bind IgG from different sources was determined. The bifunctional chimaeric protein can bind up to 23.4 mg/ml human IgG at a ratio of 1 mol of ProtA–CBD/2 mol of human IgG, and can purify up to 11.6 mg/ml rabbit IgG from a serum. The ability to bind functionally active CBD‐affinity reagents to cellulosic microtitre plates was demonstrated. Our results indicate that a combination of CBD‐affinity reagents and cellulosic microtitre plates is an attractive diagnostics matrix for the following reasons: (i) cellulose exhibits very low non‐specific binding; and (ii) CBD‐fusion proteins bind directly to cellulose at high density. A unique signal‐amplification method was developed based on the ability of ProtA–CBD to link stained cellulose particles to primary antibody in a Western blot.

Cellulose beads bound to cellulose binding domain-fused recombinant proteins; an adjuvant system for parenteral vaccination of fish.

A recombinant form of the outer membrane protein (A-layer protein) associated with atypical Aeromonas salmonicida was expressed, fused to a cellulose binding domain (CBD) isolated from Clostridium cellulovorans. The resultant chimerical protein was bound to either Sigmacell 20((R)) or Orbicell cellulose particles. Common goldfish were injected intraperitoneally with the cellulose-protein complex and blood serum antibody levels produced against A-protein were examined weekly by means of ELISA. These titers were compared to those induced by immunization of goldfish with the same protein, with or without Freunds incomplete adjuvant, as well as to a standard bacterin-adjuvant system. Small Orbicell beads (1-10 microM) induced antibody levels that were equal to the titers produced by the adjuvanted protein and bacterin formulae. In comparison, the larger Sigmacell particles (10-20 microM) proved to be poor immunopotentiators. The long-term titer elicited from a single injection of A-protein bound to Orbicell beads was equivalent to that induced by two injections. All the vaccinated fish demonstrated memory to the A-layer protein after exposure to a pathogenic load of atypical A. salmonicida with Orbicell treated fish displaying the highest titer. No direct correlation was found between the presence of anti-A-protein antibodies and protection against infection. The paper describes a simple and safe method to increase the potential immunogenicity of soluble recombinant proteins by employing relatively inexpensive cellulose particles.

Expression of Arabidopsis Thaliana Endo-1,4-ß-Glucanase (cel1) in Transgenic Poplar Plants

The isolation of an elongation-specific endo-l,4-s-glucanase-cel1 from Arabidopsis thaliana was made possible by the fact that considerable homology exists between different endo-1,4-s-glucanase (EGase) genes from different plants. The A. thaliana cel1 cDNA gene was cloned and found to encode a 54-kDa protein. Northern blot analysis of cel1 suggested its developmental regulation. RNA transcripts were undetectable in fully expanded leaves as well as at the basal internode of flowering stems. However, a strong transcript signal was detected in the elongating zone of flowering stems. Transgenic poplar plants expressing cel1 gene under CaMV 35S promoter, had significantly longer internodes as well as longer fiber cells. These results further substantiate the link between cel1 expression and plant cell elongation.

Expression, cross-linking and characterization of recombinant chitin binding resilin

Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance protein-carbohydrate composite material. We describe for the first time a comprehensive study on the mechanical, structural and biochemical function of chitin binding recombinant Drosophila melanogaster resilin. Various resilin constructs were cloned including the full length gene enabling Ni-NTA purification, as well as heat and salt precipitation for rapid and efficient purification. The binding isotherms and constants (K d , B max ) of resilin to chitin via its chitin binding domain were determined and displayed high affinity to chitin, implying its important role in the assembly of the resilin-chitin composite. The structural and elastic properties were investigated using Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and Atomic Force Microscopy (AFM) with peroxidase crosslinked solid resilin materials. Generally, little structural organization was found by these biophysical methods, suggesting structural order was not induced by the dityrosine crosslinks. Further, the elastomeric properties found from the full length protein compared favorably with the shorter resilin generated previously from exon 1. The unusual elastomeric behavior of this protein suggests possible utility in biomaterials applications.