Countercurrent dialysis cartages can also be used, in which the solution to be dialyzed flow in one direction, and the dialysate in the opposite direction outside of the membrane. Similarly, ultrafiltration membranes, which are made from cellulose acetate or other porous materials, can be used to purify and concentrate an enzyme larger than certain molecular weight.
The molecular weight is called the molecular weight cutoff and is available in a large range from different membranes. The ultrafiltration process is usually carried out in a cartridge loaded with the enzyme to be purified. Centrifugal force or vacuum is applied to accelerate the process. Both dialysis and ultrafiltration are quick but somewhat vague on distinguishing the molecular weight, whereas size exclusion chromatography gives fine fractionation from the raw mixture, allowing separation of the desired enzyme from not only small molecules but also other enzymes and proteins.
Size exclusion chromatography, also known as gel-filtration chromatography, relies on polymer beads with defined pore sizes that let particles smaller than a certain size into the bead, thus retarding their egress from a column. In general, the smaller the molecule, the slower it comes out of the column. Other factors including the pore size, protein shape, column volumes, and ionic strength of the eluent could also change the result of purification. Figure 2. The scheme of dialysis.
Enzyme molecules red dots are retained in the dialysis bag and separated from other smaller molecules blue dots. Like other proteins, enzymes can be separated on the basis of polarity, more specifically, their net charge, charge density, and hydrophobic interactions. In ion-exchange chromatography, a column of beads containing negatively or positively charged functional groups are used to separate enzymes. The cationic enzymes can be separated on anionic columns, and anionic enzymes on cationic column.
Electrophoresis is a procedure that uses an electrical field to cause permeation of ions through a solid or semi-solid matrix or surface resulting in separations on constituents on the basis of charge density. The distance a protein migrates in SDS-PAGE is inversely proportional to the log of its molecular radius, which is roughly proportional to molecular weight. Similarly, a matrix with gradient pH can be used in isoelectric focusing separation.
The matrix used can be liquid or a gel poured into either a cylindrical shape, or a flat plate. Hydrophobic interaction chromatography HIC employs hydrophobic interactions to distinguish different enzymes, which are adsorbed on matrices such as octyl- or phenyl-Sepharose. A gradient of decreasing ionic strength, or possibly increasing non-polar solvent concentration can be used to elute the proteins, giving fractions that usually contain relatively high-pure enzymes.
High-pressure liquid chromatography HPLC uses the same principle of separation of HIC, which is filled with more finely divided and tuned materials and thus allows more choices of eluents and results in better separation. Note that HPLC could be based on polarity, affinity, or both. Affinity chromatography is another powerful and generally applicable means of purifying enzymes.
This technique takes advantage of the high affinity of many enzymes for specific chemical groups. In general, affinity chromatography can be effectively used to isolate a protein that recognizes a certain group by 1 covalently attaching this group or a derivative of it to a column, 2 adding a mixture of proteins to this column, which is then washed with buffer to remove unbound proteins, and 3 eluting the desired protein by adding a high concentration of a soluble form of the affinity group or altering the conditions to decrease binding affinity.
Affinity chromatography is most effective when the interaction of the enzyme and the molecule that is used as the bait is highly specific. A special example of ligand-affinity chromatography is the Ni-NTA nickel — nitrolotriacetic acid-agaraose affinity chromatography.
This ligand binds tightly to a 6 amino acid peptide consisting only of histidines His6. This allows the affinity-purification of such a protein using Ni-NTA without having to design a special ligand-affinity column. Look at the literature on similar proteins and see what other people did.
If that is not an option, then you may need to investigate different columns. However, you will need an assay to see where your protein elutes. One of the possible steps you may encounter in purification is to precipitate either your enzyme of interest, or the contaminating proteins.
Concentrating your enzyme may, in fact, be important following chromatography. If your enzyme ends up being spread across several elution fractions, then the decreased concentration will result in lower activity and require higher volumes for assays. Concentration may be necessary to squeeze more enzyme into lower volumes. How exactly are you going to assay your enzyme? Of course, often, this will not be the case. For one thing, just finding a biochemical reaction where substrates and products have different wavelengths can be difficult enough.
Your primary reaction may not have any wavelength change, but your secondary reaction will. For example:. One of the enzymes I worked with, creatine kinase, took not just one but two coupling steps to give me a chromophore that I could work with. However, the substrates for the coupling enzyme, not to mention those commercial coupling enzymes themselves, got to be expensive.
Has this helped you? Then please share with your network. In order to verify your enzyme at different pH, you have to work with different buffers. In your case, I suppose that your enzyme is in a culture medium growth medium of the strain that produced it. If you need to test several conditions I recommend a DoE approach.
It gives better results and helps to improve faster than rational experiments. Band of interest protein ChBD-GFP disappeared in cell lysate after binding lane 2 when compared with cell lysate before binding lane 1 , the majority of interest protein was retained in precipitate lane 3.
Figure 8. Stability analysis of immobilized and free LOGX. A pH stability. B Thermal stability. Immobilized enzyme showed much better stability than free enzyme. We speculated that the yield of repeating enzymatic conversion decreased may due to the loss enzyme activity, which was consistent with the results of temperature stability Figure 8B. Figure 9. Experiments show that this method is feasible as expected.
This paper described the construction of a universal fusion vectors pETChBD-X allowing expression of fusion proteins which can be affinity purified by a one-step procedure. These conditions are very advantageous for recombinant protein purification. Firstly, chitin is a kind of absolutely non-polluting material as it is natural. Secondly, the time, cooling and reagent costs will be further compressed by optimization. Generally speaking, many reported cases of protein purification based on carbohydrate-binding domain usually need overnight incubating such as Myung et al.
For example, Ramirez et al. Thirdly, the whole process of purification or immobilization is simple, only incubation and centrifugation are needed. By all accounts, the protein purification and immobilization method provided in this study is suitable for large-scale industrial application because of its unique advantages. The visualization process intuitively reflects these steps and SDS-AGE analysis confirmed the high-purity of interest protein.
Another recombinant protein ChBD-LGOX was successfully purified and immobilized in single step which revealed the convenience of this method. Moreover, the stability of enzyme was significantly enhanced after immobilization. This is consistent with some reported cases, for example, the cis -epoxysuccinate hydrolases fused with cellulose-binding domain and immobilized on cellulose showed better stability than its wild-type version Cui et al.
The characterization of ChBD-LGOX changed especially the enzymatic activity decreased by a big margin which revealed an obvious disadvantage of this immobilization approach. Stereospecific blockade between fusion tag and target protein might be the main reason for this phenomenon, further research is needed to overcome this shortage. Although the yield of repeating enzymatic conversion decreased by a big margin, this is mainly due to the relatively poor stability of the enzyme itself.
In summary, the ChBD-AB affinity tag system described here provides a novel tool for an innovative alternative method for purifying, immobilizing, and spreading fusion proteins. Great advantages including low cost, no pollution, easy operation, rapidness, and high-purity suggested that the plasmid vector pETChBD-X has potential value in immobilization and purification of enzymes.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. JZ was responsible for experimental design and thesis writing. JC and NZ were responsible for experimental operation. AZ and KC provided experimental materials. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Boraston, A. Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Campion, E. Walls and S. Google Scholar. Cha, J. Purification-free, target-selective immobilization of a protein from cell lysates. Chern, J. Chitin-binding domain based immobilization of D-hydantoinase. Coskun, O. Separation techniques: chromatography. Cui, G. High yield recombinant expression, characterization and homology modeling of two types of cis-epoxysuccinic acid hydrolases.
Protien J. Demishtein, A. Characterization of a dockerin-based affinity tag: application for purification of a broad variety of target proteins. Ferrandon, S. A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag.
Acta , 31— Hashimoto, M. Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL Inokuma, K. Direct ethanol production from N-acetylglucosamine and chitin substrates by Mucor species. Jin, J. The use of affinity tags to overcome obstacles in recombinant protein expression and purification. Protien Pept. Leister, T. Li, Z. Affinity monolith chromatography: a review of general principles and applications.
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