Purification of trypsin by affinity precipitation and combining with aqueous two-phase extraction

Summary Soybean trypsin inhibitor was bound to a pH-sensitive reversibly soluble-insoluble polymer (AS-L) for affinity precipitation of trypsin. Trypsin was purified from a crude extract 5.4-fold and with 61% yield, however, the reusability of the ligand was poor. This can be overcome by combining aqueous two-phase extraction with affinity precipitation, which improved trypsin yield and purity factor after repeated ligand usage.     

Polymerized liposome as ligand carrier for affinity precipitation of proteins

A polymerized liposome (PLS) was prepared using a synthesized phospholipid with a diacetylene moiety in the hydrophobic chain and an amino group in the hydrophilic head. The PLS was used as a novel ligand carrier for affinity precipitation of proteins because it showed a reversibly precipitable property on salt addition and removal. Soybean trypsin inhibitor (STI) was easily immobilized on the PLS by a one-step carbodiimide reaction. The PLS showed no nonspecific adsoprtion of proteins. It had a large ligand coupling capacity, and then a large adsorption capacity for trypsin after STI immobilization. The PLS with immpbilized STI was recycled three times for the purification of trypsin from a crude pancreatic extract. Although the degree of purification was compromised by the impurity of the STI employed, in each run the purification factor reached about 6 and more than 80% of trypsin activity was recovered. The results indicated that the PLS was a potential ligand carrier for affinity precipitation of proteins. (c) 1995 John Wiley & Sons, Inc.     

Affinity precipitation of proteins: design criteria for an efficient polymer

Affinity precipitation is fast emerging as a successful technique for the purification of proteins which can be introduced at an early stage of downstream processing. The technique applies the use of reversibly soluble-insoluble polymers which have either natural or synthetic origin. Apart from the successful use of some natural polymers, such as chitosan and alginate, the vast application of the technique depends upon the design of efficient synthetic polymers. In this laboratory, N-isopropylacrylamide (NIPAM) copolymers have been developed for metal chelate affinity precipitation of proteins. The copolymers of 1-vinylimidazole (VI) and iminodiacetic acid (IDA) with NIPAM were synthesized. The copolymers were thoroughly characterized with a view to designing an efficient soluble-insoluble polymer for metal chelate affinity precipitation of proteins.     

Production of cellobiose by enzymatic hydrolysis: removal of beta-glucosidase from cellulase by affinity precipitation using chitosan

Removal of beta-glucosidase (BG) from cellulase is essential to the enzymatic production of cellobiose from cellulose because of the high reactivity of BG with cellobiose to form glucose. Chitosan is a reversibly soluble-insoluble polymer depending on pH, and it has an affinity with the other components, endo-beta-1,4-glucanase and cellobiohydrolase, or cellulase. The affinity precipitation technique using chitosan is an effective way to fractionate cellulase for the above purpose. Hydrolysis experiments of cellulose with the residual fractionated enzyme gave higher cellobiose contents in the soluble sugar products. (c) 1993 John Wiley & Sons, Inc.     

Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.     

Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads

Silica gel bead coated with macroporous chitosan layer (CTS-SiO₂) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu²⁺, Zn²⁺, Ni²⁺ ions, respectively on CTS-SiO₂, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu²⁺-CTS-SiO₂ with the value of 4980 ± 125 IU g⁻¹ of the adsorbent, while the maximal adsorption capacity for trypsin on Zn²⁺ and Ni²⁺ loaded adsorbent was 3762 ± 68 IU g⁻¹ and 2636 ± 53 IU g⁻¹, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO₂ beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO₂ could effectively digest BSA revealed by HPLC peptide mapping.     

Free polymeric bioligands in aqueous two-phase affinity extractions of microbial xylanases and pullulanase

Two reversibly soluble-insoluble polymers (viz. Eudragit S-100 and alginate) were used as free macroaffinity bioligands in polyethylene glycol (PEG)/salt two-phase systems for separation of enzymes. Incorporation of Eudragit S-100 and alginate in the PEG phase led to considerable selectivity in separation of microbial xylanases and pullulanase, respectively. Xylanase from Aspergillus niger was recovered 93% with 56-fold purification, whereas the enzyme from Trichoderma reesei and Bacillus amyloliquefaciens was obtained with 93% activity recovery (31-fold purification) and 90% activity recovery (32-fold purification), respectively. From Bacillus acidopullulyticus pullulanase, 85% enzyme activity recovery with 44-fold purification was obtained. The approach described here shows the potential of developing into a general approach for use of reversibly soluble-insoluble macroaffinity ligand in two-phase affinity extraction.     

Immobilized trypsin on hydrophobic cellulose decorated nanoparticles shows good stability and reusability for protein digestion

The preparation of biocatalysts based on immobilized trypsin is of great importance for both proteomic research and industrial applications. Here, we have developed a facile method to immobilize trypsin on hydrophobic cellulose-coated silica nanoparticles by surface adsorption. The immobilization conditions for the trypsin enzyme were optimized. The as-prepared biocatalyst was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and elemental analysis. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and denaturants. In addition, the immobilized trypsin showed good durability for multiple recycling. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of two widely used protein substrates: bovine serum albumin (BSA) and cytochrome c. The surface adsorption protocols for trypsin immobilization may provide a promising strategy for enzyme immobilization in general, with great potential for a range of applications in proteomic studies.     

The continuous isolation of trypsin by affinity adsorbent recycling

A method for the continuous affinity separation of proteins is described in which the adsorbent, in the form of a polymer belt, is recycled through feedstock and eluent liquid flows. As the belt is nonporous, contact between the solute and the ligand is not diffusion-dependent. Consequently, rapid cycle rates are possible. Soybean trypsin inhibitor immobilized on nylon was used as an affinity ligand for the isolation of trypsin. During a 30-h continuous run, trypsin was isolated from a crude preparation of bovine pancreas with a recovery of 30% to 40%. Approximately 18 mg of trypsin was obtained from 500 mg of protein using a total of approximately 10 mug of ligand. Electrophoretic analysis of the eluent showed that chymotrypsin, which also binds to SBTI, was the only major contaminant of the product. It was demonstrated that the highest rates of protein purification were obtained using solid/liquid contact times well below that required to achieve saturation of the affinity adsorbent. Slower adsorbent recycle rates, which achieved higher protein binding per unit area of belt, resulted in lower protein purification per unit time. The rate of purification was also dependent on the concentration of target protein in the adsorption chamber at steady state. As high concentrations increased losses from the chamber outflow, this resulted in a compromise between throughput and recovery during the adsorption phase. Under the conditions investigated, recoveries of over 60% were obtained, and a maximum throughput of approximately 2.5 mg trypsin per hour was achieved. Preliminary studies have shown that this can be improved by compartmentalizing the adsorption chamber, which can reduce losses from the adsorption chamber to less than 5%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 538-545, 1997.     

Simultaneous refolding/purification of xylanase with a microwave treated smart polymer

Affinity precipitation with a smart polymer, Eudragit S-100 (a methyl methacrylate polymer), was exploited for simultaneous refolding and purification of xylanase. Affinity precipitation consisted of this reversibly soluble-insoluble polymer-binding xylanase selectively. The complex was precipitated by lowering the pH and xylanase was eluted off the polymer using 1 M NaCl. For refolding experiments, the commercial preparation of Aspergillus niger xylanase was denatured with 8 M urea. Addition of microwave irradiated Eudragit S-100 and affinity precipitation led to recovery of 96% enzyme activity by refolding. Simultaneously, the enzyme was purified 45 times. Thermally inactivated preparation, when subjected to similar steps, led to 95% recovery of enzyme activity with 42-fold purification. The strategy has the potential for recovering pure proteins in active forms from overexpressed proteins, which generally form inclusion bodies in E. coli.     

Improvement of cell lysis activity of immobilized lysozyme with reversibly soluble-insoluble polymer as carrier

Summary Hen egg white lysozyme was immobilized by covalent binding to a polymer showing reversibly soluble-insoluble characteristics with pH change. The retention of the specific activity of the immobilized enzyme can be as high as 41% of that of the free enzyme. The immobilized enzyme could be used in repeated batch lysis of M. lysodeikticus cells and to enhance the release of intracellular proteins 1.4 folds when compared with batch operation.     

Highly stable trypsin‐aggregate coatings on polymer nanofibers for repeated protein digestion

A stable and robust trypsin‐based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300‐fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC‐MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in “real‐world” proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.     

The amino acid sequence and reactive site of a single-headed trypsin inhibitor from wheat endosperm

The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion withStaphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.     

An activated medium with high durability and low nonspecific adsorption: application to protein A chromatography

Activated media allow the user to easily synthesize a variety of affinity media. We have developed a novel activated medium based on porous silica modified with phosphorylcholine (PC) and N-hydroxysuccinimide (NHS) groups for the purpose of high-throughput purification and reducing nonspecific protein adsorption. The PC groups function as suppressors of nonspecific protein adsorption, whereas the NHS groups are able to covalently bind to the primary amino groups of ligands. Because protein A affinity medium is the most frequently used affinity medium, we prepared protein A media in which a recombinant protein A was bound to the NHS groups of the activated media and evaluated its utility. After optimizing various factors in the synthetic process, the resultant protein A medium showed improved durability at a high flow rate over 300 purification cycles and reduced nonspecific protein adsorption compared with commercially available protein A media.     

Precipitation with poly acrylic acid as a trypsin bioseparation strategy

Precipitation of enzymes with reversible soluble–insoluble polymers is a simple approach which can be easily scaled up. This work reports investigations aiming at verifying the existence of specific interactions and complex formation between porcine trypsin and poly acrylic acids using spectroscopy techniques. The trypsin–polymer complex was insoluble at pH lower than 5, with a stoichiometric ratio polymer mol per protein mol of 1:148. It took only a minute for the insoluble complex to form and it was redissolved modifying the pH of the medium. The enzymatic activity of trypsin was maintained even in the presence of the polymer and after precipitation poly acrylic acid presence protect the enzyme from itself degradation. The conditions of complex formation were studied using pure proteins that could be applied on porcine pancreas homogenates as an isolation strategy of trypsin.     

Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversibly soluble polymer Eudragit(TM) L-100

The non-covalent immobilization of a commercial preparation of xylanase from A. niger was carried out on a reversibly soluble-insoluble enteric polymer Eudragit(TM) L-100. The immobilization of the xylanase activity by adsorption was simultaneously accompanied by removal of cellulase activity since the latter did not bind to the polymer. Thus, the soluble enzyme derivative may be useful for treatment of paper pulp bleaching in paper industry. The immobilized xylanase retained 60% of its activity toward xylan as the substrate. No change was observed in the pH optimum (5.5) of the enzyme upon immobilization. Only marginal increase in the K(m) of the free enzyme (3.6 mg ml(-1) to 5.0 mg ml(-1)) upon immobilization on the soluble polymer reflected that the enzyme-substrate binding continues to be efficient in spite of the macromolecular nature of the substrate. Fluorescence spectroscopy and UV difference spectroscopy were used to probe the change(s) in the enzyme structure upon immobilization. This change in structure was correlated with the "effectiveness factor" of the enzyme activity. CD spectra also showed that the enzyme undergoes drastic changes in the structure.     

Analysis of the interactions between Eudragit® L100 and porcine pancreatic trypsin by calorimetric techniques

Flexible-chain polymers with charge (polyelectrolytes) can interact with globular proteins with a net charge opposite to the charge of the polymers forming insoluble complexes polymer-protein. In this work, the interaction between the basic protein trypsin and the anionic polyelectrolyte Eudragit^® L100 was studied by using isothermal calorimetric titrations and differential scanning calorimetry. Turbidimetric assays allowed determining that protein-polymer complex was insoluble at pH below 5 and the trypsin and Eudragit^® L100 concentrations required forming the insoluble complex. DSC measurements showed that the T_m and denaturalization heat of trypsin increased in the polymer presence and the complex unfolded according to a two-state model. ΔH° and ΔS° binding parameters obtained by ITC were positives agree with hydrophobic interaction between trypsin and polymer. However, ionic strength of 1.0 M modified the insoluble complex formation. We propose a mechanism of interaction between Eudragit^® L100 and trypsin molecules that involves both hydrophobic and electrostatic interactions. Kinetic studies of complex formation showed that the interaction requires less than 1 min achieving the maximum quantity of complex. Finally, a high percentage of active trypsin was precipitated (approximately 76% of the total mass of protein). These findings could be useful in different protocols such as a protein isolation strategy, immobilization or purification of a target protein.     

Nanoneedle Surface Modification with 2-Methacryloyloxyethyl Phosphorylcholine Polymer to Reduce Nonspecific Protein Adsorption in a Living Cell

Previously, we developed a new molecular delivery system to target single living cells by using atomic force microscope and ultrathin needle referred to as nanoneedle. This system delivers molecules into the cell by attaching them to the surface of nanoneedle. However, nonspecific protein adsorption on the nanoneedle surface inside the living cells limits the range of application of this system. In the present study, we focused on nonspecific protein adsorption onto the nanoneedle surface inside the cells and examined whether this protein adsorption was reduced by modifying the nanoneedle surface with a biocompatible phospholipid polymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC) unit. MPC polymer coating of the surface of silicon wafer reduced nonspecific adsorption of proteins from liver extracts and prevented the formation of clot-like protein aggregates. MPC polymer also decreased nonspecific adsorption of cytosolic protein onto the nanoneedle surface inside the living cell. On the other hand, MPC polymer showed no effect on nonspecific mechanical interaction between nanoneedle and the cell components. Surface modification with MPC polymer is a useful technique to modify the surface properties of nanoneedle.     

胰蛋白酶抑制物对尼罗非鲫生长的影响

本实验研究了生、熟大豆中含有的胰蛋白酶抑制物(TI)对尼罗非鲫生长的影响。实验结果表明:沸水热处理不仅使生大豆中81.4％的TI失去活性,而且还显著地提高了大豆蛋白质系数,PER从1.12提高到1.76。当颗粒饲料的Tl含量低于0.9mg/g时,尼罗非鲫的生长正常;但高于此量吋,其生长速度明显降低。       

Affinophoresis of trypsins with an anionic affinophore

Affinophoresis (Shimura, K. and Kasai, K. (1982) J. Biochem. 92, 1615–1622) is a newly devised electrophoretic separation technique for biomolecules, using an affinophore. The affinophore is a macromolecular polyelectrolyte bearing affinity ligands. It migrates rapidly in an electric field, and molecules which have affinity for the ligand are carried with it and separated from other molecules. An anionic affinophore for trypsin was synthesized. p-Aminobenzamidine, a competitive inhibitor of trypsin, was coupled to one-fifth of the car?yl groups of polyacrylyl-β-alanyl-β-alanine by the use of water-soluble carbodiimide and the residual car?yls were converted to sulfonate groups by coupling with aminomethanesulfonic acid. Affinophoresis was carried out in 1% agarose gel plates, and the protein bands were detected with Coomassie brilliant blue R250. Enhanced migrations of bovine and Streptomyces griseus trypsins towards the anode were observed with the anionic affinophore. The migrations of inactive forms prepared by active site modifications were scarcely affected. However, the affinophore was not effective for Streptomyces erythreus trypsin, an anionic trypsin, probably because of ionic repulsion between the anionic molecules. S. griseus trypsin was separated from Pronase by affinophoresis.