Liquid crystal elastomer foams with elastic properties specifically engineered as biodegradable brain tissue scaffolds.

Tissue regeneration requires 3-dimensional (3D) smart materials as scaffolds to promote transport of nutrients. To mimic mechanical properties of extracellular matrices, biocompatible polymers have been widely studied and a diverse range of 3D scaffolds have been produced. We propose the use of responsive polymeric materials to create dynamic substrates for cell culture, which goes beyond designing only a physical static 3D scaffold. Here, we demonstrated that lactone- and lactide-based star block-copolymers (SBCs), where a liquid crystal (LC) moiety has been attached as a side-group, can be crosslinked to obtain Liquid Crystal Elastomers (LCEs) with a porous architecture using a salt-leaching method to promote cell infiltration. The obtained SmA LCE-based fully interconnected-porous foams exhibit a Young modulus of 0.23 ± 0.07 MPa and a biodegradability rate of around 20% after 15 weeks both of which are optimized to mimic native environments. We present cell culture results showing growth and proliferation of neurons on the scaffold after four weeks. This research provides a new platform to analyse LCE scaffold-cell interactions where the presence of liquid crystal moieties promotes cell alignment paving the way for a stimulated brain-like tissue.

Recovery of bovine lysozyme from transgenic sugarcane stalks: extraction, membrane filtration, and purification.

Increased industrial use of sugarcane (Saccharum spp. hybrid) for food and bioenergy has led to considerable improvements in its genetic transformation, which allowed the development of not only pest- and herbicide-resistant lines but also lines expressing high-value bioproducts and biopolymers. However, the economic benefits of using inexpensive transgenic plant systems for the production of industrial proteins could be offset by high downstream processing costs. In this work, transgenic sugarcane expressing recombinant bovine lysozyme (BvLz) was used to evaluate the feasibility of extraction and fractionation of recombinant proteins expressed in sugarcane stalks. Three pH levels (4.5, 6.0 and 7.5) and three salt concentrations (0, 50, and 150 mM NaCl) were tested to determine BvLz and total protein extractability. Two extraction conditions were selected to prepare BvLz extracts for further processing by cross-flow filtration, a suitable method for concentration and conditioning of extracts for direct applications or prior to chromatography. Partial removal of native proteins was achieved using a 100 kDa membrane but 20-30 % of the extracted BvLz was lost. Concentration of clarified extracts using a 3 kDa membrane resulted in twofold purification and 65 % recovery of BvLz. Loading of concentrated sugarcane extract on hydrophobic interaction chromatography (HIC) resulted in 50 % BvLz purity and 69 % recovery of BvLz.

Effect of processing on the recovery of recombinant beta-glucuronidase (rGUS) from transgenic canola.

This study addresses the processing of transgenic canola seed for production of recombinant proteins by using beta-glucuronidase (rGUS) as a model protein. The major processing steps that were investigated included dry and wet grinding of the seed, solvent extraction of canola oil, and protein extraction. rGUS in canola seed was stable for at least 2 weeks of incubation at 38 degrees C and for more than 5 months at 10 degrees C. At 70 degrees C, the residual activity changed inversely to the initial moisture content of the seed. The comparison of wet and dry processing revealed no significant differences in protein recovery. rGUS was stable during the defatting of transgenic canola flakes with hexane at 66 degrees C, whereas 2-propanol extraction at the same temperature reduced the extractable enzyme activity by almost 50%. The particle size of the ground seed was important for the extraction efficiency. A faster extraction and greater protein yield was achieved by extracting particles with an average diameter equal to or smaller than 255 microm. More than 80% rGUS was extracted in one stage with sodium phosphate buffer of pH 7.5.

Suppression of electromyogram interference on the electrocardiogram by transform domain denoising.

A method for suppression of electromyogram (EMG) interference in electrocardiogram (ECG) recordings is presented. By assuming that the EMG is long-term non-stationary Gaussian noise, two successive decompositions were proposed, and the data transformed for Wiener filtering. Successive ECG cycles were rearranged and aligned by the R-wave, forming a matrix containing separated heart cycles in its rows. A short-window discrete cosine transform (DCT) was applied to the columns of the matrix for inter-cycle de-correlation. Next, Wiener filtering in a translation-invariant wavelet domain was performed on the DCT-transformed matrix rows for de-correlation of the data into each ECG cycle. The method resulted in an improvement in the signal-to-noise ratio of more than 10 dB, a threefold reduction in mean relative amplitude errors and reduced ripple artifacts around the signal transients, thus preserving the waveform in diagnostically important signal segments.

Molecular farming of industrial proteins from transgenic maize.

Recombinant egg white avidin and bacterial B-glucuronidase (GUS) from transgenic maize have been commercially produced. High levels of expression were obtained in seed by employing the ubiquitin promoter from maize. The recombinant proteins had activities that were indistinguishable from their native counterparts. We have illustrated that down-stream activities in the production of these recombinant proteins, such as stabilizing the germplasm and processing for purification, were accomplished without any major obstacles. Avidin (A8706) and GUS (G2035) are currently marketed by Sigma Chemical Co.

Effects of mutations in the starch-binding domain of Bacillus macerans cyclodextrin glycosyltransferase.

Cyclodextrin glycosyltransferase (CGTase) is an industrially important enzyme that produces cyclodextrins (CD) from starch by intramolecular transglycosylation. CGTase consists of five globular domains labeled A through E. To better understand the role of domain E in CGTase catalysis, we have constructed several mutants of Bacillus macerans CGTase. Removing the entire E domain resulted in an inactive enzyme. Adding six amino acids between domains D and E caused a decrease in activity and thermostability. Replacing domain E with the similar starch-binding domain from Aspergillus awamori glucoamylase I caused a drastic decrease in activity, indicating the necessity of correct alignment of bound substrate. Substituting tyrosine residue 634 (Tyr634) with phenylalanine had very little effect on activity or thermostability. Substituting Tyr634 with glycine resulted in a 25% increase of specific cyclization and starch-hydrolyzing activities compared with that of the wild-type enzyme. The latter mutant was less thermostable. The results of this study indicate that domain E is important for the stability and integrity of B. macerans CGTase.

Production and purification of two recombinant proteins from transgenic corn.

This study reports the production, purification, and characterization of recombinant Escherichia coli beta-glucuronidase (GUS) and chicken egg-white avidin from transgenic corn seed. The avidin and gus genes were stably integrated in the genome and expressed over seven generations. The accumulation levels of avidin and GUS in corn kernel were 5.7% and 0.7% of extractable protein, respectively. Within the kernel, avidin and GUS accumulation was mainly localized to the germ, indicating possible tissue preference of the ubiquitin promoter. The storage-stability studies demonstrated that processed transgenic seed containing GUS or avidin can be stored at 10 degrees C for at least 3 months and at 25 degrees C for up to 2 weeks without a significant loss of activity. The heat-stability experiments indicated that GUS and avidin in the whole kernels were stable at 50 degrees C for up to 1 week. The buffer composition also had an affect on the aqueous extraction of avidin and GUS from ground kernels. Avidin was purified in one step by using 2-iminobiotin agarose, whereas GUS was purified in four steps consisting of adsorption, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Biochemical properties of purified avidin and GUS were similar to those of the respective native proteins.

Processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase.

The tools of plant biotechnology that have been developed to improve agronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry. This study addresses several processing and protein recovery issues that are relevant to utilizing transgenic corn as a protein production system. The gus gene coding for beta-glucuronidase (rGUS) was stably integrated and expressed over four generations. The accumulation level of rGUS reached 0.4% of total extractable protein. Within the kernel, rGUS was preferentially accumulated in the germ even though a constitutive ubiquitin promoter was used to direct gus expression. Fourth-generation transgenic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS. Transgenic seed containing rGUS could be stored at an ambient temperature for up to two weeks and for at least three months at 10 degrees C without a significant loss of enzyme activity. rGUS exposed to dry heat was more stable in ground than in whole kernels. The enzyme stability was correlated with the moisture loss of the samples during the heating. Transgenic seed was dry-milled, fractionated, and hexane extracted to produce full-fat and defatted germ fractions. The results of the aqueous extraction of rGUS from ground kernels, full-fat germ, and defatted-germ samples revealed that approximately 10 times more rGUS per gram of solids could be extracted from the ground full-fat germ and defatted-germ than from the kernel samples. The extraction of corn oil from ground germ with hot hexane (60 degrees C) did not affect the extractable rGUS activity. rGUS was purified from ground kernels and full-fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography. Similar purity and yield of rGUS were obtained from both extracts. Biochemical properties of rGUS purified from transgenic corn seed were similar to those of E. coli GUS.

Production of recombinant proteins in transgenic plants: Practical considerations.

This review is based on our recent experience in producing the first commercial recombinant proteins in transgenic plants. We bring forward the issues that have to be considered in the process of selecting and developing a winning transgenic plant production system. From the production point of view, transcription, posttranscription, translation, and posttranslation are important events that can affect the quality and quantity of the final product. Understanding the rules of gene expression is required to develop sound strategies for optimization of recombinant protein production in plants. The level of recombinant protein accumulation is critical, but other factors such as crop selection, handling and processing of transgenic plant material, and downstream processing are equally important when considering commercial production. In some instances, the cost of downstream processing alone may determine the economic viability of a particular plant system. Some of the potential advantages of a plant production system such as the high levels of accumulation of recombinant proteins, glycosylation, compartmentalization within the cell, and natural storage stability in certain organs are incentives for aggressively pursuing recombinant protein production in plants. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 473-484, 1997.

Deletion analysis of the starch-binding domain of Aspergillus glucoamylase.

The large form of glucoamylase (GAI) from Aspergillus awamori (EC 3.2.1.3) binds strongly to native granular starch, whereas a truncated form (GAII) which lacks 103 C-terminal residues, does not. This C-terminal region, conserved among fungal glucoamylases and other starch-degrading enzymes, is part of an independent starch-binding domain (SBD). To investigate the SBD boundaries and the function of conserved residues in two putative substrate-binding sites, five gluco-amylase mutants were constructed with extensive deletions in this region for expression in Saccharomyces cerevisiae. Progressive loss of both starch-binding and starch-hydrolytic activity occurred upon removal of eight and 25 C-terminal amino acid residues, or 21 and 52 residues close to the N-terminus, confirming the requirement for the entire region in formation of a functional SBD. C-terminal deletions strongly impaired SBD function, suggesting a more important role for one of the putative binding sites. A GAII phenocopy showed a nearly complete loss of starch-binding and starch-hydrolytic activity. The deletions did not affect enzyme activity on soluble starch or thermo-stability of the enzyme, confirming the independence of the catalytic domain from the SBD.

Domain E of Bacillus macerans cyclodextrin glucanotransferase: An independent starch-binding domain.

The starch-binding domains of glucoamylase I (SBD of GA-I) from Aspergillus awamori and of cyclodextrin glucanotransferase (domain E of CGTase) from Bacillus macerans were fused to the C-terminus of beta-galactosidase (beta-gal) The majority of the fusion proteins produced in Escherichia coli were found as inclusion bodies. Active fusion proteins were purified by partial solubilization of the inclusion bodies with 2 M urea followed by affinity chromatography. Adsorption isotherms of purified fusion proteins on corn starch and cross-linked amylose were generated. The beta-gal fusion proteins had similar affinities for cross-linked amylose and corn starch but significantly different saturation capacities on corn starch. The adsorption and elution data from the potato starch column as well as the adsorption isotherms of p-gal-domain E fusion protein (BDE109) on corn starch and cross-linked amylose demonstrated that domain E of CGTase is an independent domain, which retained its starch-binding activity when separated from the other four (A-D) domains in CGTase. (c) 1995 John Wiley & Sons Inc.

Starch-binding domain of Aspergillus glucoamylase-I. Interaction with beta-cyclodextrin and maltoheptaose.

The characterization is reported of two peptide fragments (SBD106 and SBD122) containing the starch-binding domain (SBD) of Aspergillus sp. glucoamylase I. The starch-binding peptides were produced in Escherichia coli as fusion proteins of the maltose-binding protein (MBP). SBD106 (11.9 kDa) and SBD122 (13.8 kDa) were purified from the factor Xa digest of MBP fusion proteins. The amino acid compositions were similar to those deduced from their amino acid sequences. The interactions of beta-cyclodextrin and maltoheptaose with purified SBD peptides were investigated by UV difference spectroscopy. SBD106 and SBD122 bound specifically beta-cyclodextrin with a dissociation constant (Kd) of 34 microM and 23.5 microM, respectively. Maltoheptaose binding to SBD106 and SBD122 was weaker than that of beta-cyclodextrin; dissociation constants were 0.57 and 0.50 mM, respectively. The results indicate that the intramolecular disulfide bonding is not required for the domain functioning and that O-glycosylation is not critical for the functioning of the starch-binding domain, but may affect its conformation and dynamics.

Functional starch-binding domain of Aspergillus glucoamylase I in Escherichia coli.

We have fused three starch-binding domains (SBD) encoding gene fragments (residues 511-616, 495-616 and 481-616) of glucoamylase I (GAI) to the 3' end of the Escherichia coli malE gene encoding maltose-binding protein (MBP). The fusion proteins were produced in E. coli and were purified by chromatography on cross-linked amylose. Factor Xa digestion of the fusion proteins resulted in the release of functional SBD fragments which were separated from MBP on the basis of their differential binding to cross-linked amylose. The amino acid (aa) composition of the purified SBD fragments agreed with the respective amino acid compositions of GAI. The sizes of the SBD fragments were 11.9, 13.8 and 15.6 kDa, respectively.

Degradation of degradable starch-polyethylene plastics in a compost environment.

The degradation performance of 11 types of commercially produced degradable starch-polyethylene plastic compost bags was evaluated in municipal yard waste compost sites at Iowa State University (Ames) and in Carroll, Dubuque, and Grinnell, Iowa. Masterbatches for plastic production were provided by Archer Daniels Midland Co. (Decatur, Ill.), St. Lawrence Starch Co. Ltd. (Mississauga, Ontario, Canada), and Fully Compounded Plastics (Decatur, Ill.). Bags differed in starch content (5 to 9%) and prooxidant additives (transition metals and a type of unsaturated vegetable oil). Chemical and photodegradation properties of each material were evaluated. Materials from St. Lawrence Starch Co. Ltd. and Fully Compounded Plastics photodegraded faster than did materials from Archer Daniels Midland Co., whereas all materials containing transition metals demonstrated rapid thermal oxidative degradation in 70 degrees C-oven (dry) and high-temperature, high-humidity (steam chamber) treatments. Each compost site was seeded with test strips (200 to 800 of each type) taped together, which were recovered periodically over an 8- to 12-month period. At each sampling date, the compost row temperature was measured (65 to 95 degrees C), the location of the recovered test strip was recorded (interior or exterior), and at least four strips were recovered for evaluation. Degradation was followed by measuring the change in polyethylene molecular weight distribution via high-temperature gel permeation chromatography. Our initial 8-month study indicated that materials recovered from the interior of the compost row demonstrated very little degradation, whereas materials recovered from the exterior degraded well. In the second-year study, however, degradation was observed in several plastic materials recovered from the interior of the compost row by month 5 at the Carroll site and almost every material by month 12 at the Grinnell site. The plastic bags collected from each community followed a similar degradation pattern. To our knowledge, this is the first scientific study demonstrating significant polyethylene degradation by these materials in a compost environment.

Improved adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding domain from Aspergillus glucoamylase.

We have previously shown (Chen et al., 1991) that a beta-galactosidase (beta-gal) fusion protein (BSB133) containing 133 amino acids (aa) from the C-terminus of Aspergillus glucoamylase (GA) adsorbs strongly to starch compared to beta-gal, due to the presence of the GA starch-binding domain. We have now made deletions at the N-terminus of this 133-aa region to test the minimal size required for starch binding of beta-gal fusion proteins. Three fusion proteins (BSB119, BSB103, and BSB80) were genetically engineered, containing 119, 103, and 80 C-terminal aa from GA, respectively. The fusion proteins were expressed in Escherichia coli and purified. Purified BSB119 adsorbed to native starch at least 2-fold more strongly than did BSB133 or fusion proteins with shorter tails. Adsorption isotherms generated over a wide range of initial concentrations indicated a 10-fold difference in the loading capacity of starch for BSB119 (36.5 mg of protein/g of starch) compared to beta-gal (3.7 mg of protein/g of starch). Adsorption constants calculated from the initial slopes of the isotherms indicated a nearly 30-fold difference in affinity to starch for BSB119 (Kad = 63 mL/g of starch) compared to beta-gal (Kad = 2.3 mL/g of starch). BSB119 in the presence of crude enzyme extracts also bound to starch with a high affinity compared to a beta-gal control. Potential applications of the starch-binding tail include enzyme immobilization to starch or recovery and purification of target proteins from crude extracts.

Adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding region of Aspergillus glucoamylase.

We have constructed and purified by affinity chromatography three beta-galactosidase (beta Gal) fusion proteins (BSB133, BSBCD8, and BGA134) containing amino acid (aa) sequences from Aspergillus glucoamylase (GA). BSB133, containing the C-terminal 133 aa of GA (aa 484-616), adhered to native starch granules with a much higher affinity (Kad = 18 ml/g starch) than a beta Gal control (Kad = 0.9 ml/g starch). Two other fusion proteins, BSBCD8 and BGA134, similar in size to BSB133, adhered to starch with a relatively low affinity (Kad = 7 ml/g starch, and Kad = 4 ml/g starch, respectively). BSBCD8 differs from BSB133 by a truncation of 8 aa at the C terminus. BGA134 contains 134 aa from an overlapping region of GA (aa 380-513). These results confirm the presence of a strong starch-binding region (SBR) included in the C-terminal 133 aa of GA and indicate that the SBR can confer starch-binding activity on a fusion protein produced in Escherichia coli. In the presence of crude soluble cell extracts, the fusion proteins adsorbed by native starch granules with an affinity similar to that of the purified enzymes. BSB133 that had been adsorbed by starch from crude extracts could be eluted at a high level of purity, similar to that achieved by affinity chromatography. These results suggest that it may be feasible to use native starch as an adsorbent for the recovery and purification of recombinant fusion proteins containing the SBR. Starch has many favorable qualities for this application: it is inexpensive, stable, nontoxic, and easy to recover by centrifugation.

Activity and thermal stability of genetically truncated forms of Aspergillus glucoamylase.

Glucoamylase (GA) from Aspergillus awamori (EC 3.2.1.3) is a secreted starch hydrolase with a large catalytic domain (aa 1-440), a starch-binding domain (aa 513-616), and a highly O-glycosylated region of 72 aa of unknown function that links the catalytic and starch-binding domains. We have genetically engineered a series of truncated forms of GA to determine how much of the highly O-glycosylated region is necessary for the activity or stability of GAII, a fully active form of the enzyme that lacks the starch-binding domain. Mutations were made by inserting stop-codon linkers into restriction sites within the coding region of the GA gene, and mutated genes were expressed in Saccharomyces cerevisiae for analysis of the truncated enzymes. Our results show that up to 30 aa from the C-terminal end of GAII can be deleted with little effect on the activity, thermal stability, or secretion of the enzyme. Further deletions resulted in diminution or loss of enzyme activity on starch plates, and loss of detectable enzyme in culture supernatants, indicating that these residues are essential for GAII function.

Subsite mapping of Aspergillus niger glucoamylases I and II with malto- and isomaltooligosaccharides.

Glucoamylase, industrially derived from Aspergillus niger, was chromatographically separated into forms I and II and purified to near homogeneity. Preparations were proved to be free of D-glucosyltransferase by electrophoretic and differential inhibition tests. Maximum rates and Michaelis constants were obtained for both glucoamylases I and II with maltooligosaccharides from maltose to maltoheptaose and with isomaltooligosaccharides from isomaltose to isomaltohexaose. Subsite maps were calculated from these kinetic data and were not significantly different for the two forms. Subsites in both forms had lower affinities for D-glucosyl residues contained in isomaltooligosaccharides than for D-glucosyl residues in maltooligosaccharides.