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Enzyme Purification by Affinity Column for Tagged Enzymes

Creative Enzymes provides custom production of recombinant enzymes for lab research and industry uses. With the strong technical background in enzyme manufacturing and molecular biological techniques, various post-production services are also available to yield pure, stable enzymes. Creative Enzymes offers reliable purification services to facilitate subsequent applications with contamination-free enzymes.

The parallel use of affinity tags with recombinant DNA techniques, allows the facile modification of proteins of interest leading to efficient identification, production, and isolation from the host system. However, protein insolubility, conformation, stability, and structural flexibility, as well as low purification yields and host cell toxicity are challenges that must be resolved when microbial hosts are used to express recombinant proteins. To address these challenges of production and purification efficiency, fusion tags are incorporated to increase expression yields and influence solubility and native folding; novel tags in combination with affinity techniques increase purification yields; and proteases result in tag removal. The most commonly used tags in enzyme purification are His-tag and GST-tag, respectively. The tagged enzymes are isolated and separated by affinity chromatography.

His-tag is a short affinity tag consisting of polyhistidine residues. It is a widely used method in purification process by immobilized metal-affinity chromatography (IMAC). The basic principle is the interaction between a transition metal ion immobilized on a matrix and specific amino acid side chains. The histidine imidazole ring can act as electron donor groups and form coordination bonds with the immobilized transition metal, therefore, the interaction is very strong. When the crude enzyme flows through the matrices, enzymes contain histidine residues are efficiently retained on IMAC column matrices. At Creative Enzymes, His-tag has been successfully used in the purification of enzymes from many expression systems. This purify approach results in 100-fold enrichments in a single purification step. The enzyme purity can be up to 95% with a high yield.

Enzyme Purification by Affinity Column for Tagged Enzymes Figure: The scheme of adding a His-tag onto the target protein using a primer that contains the tag.

Glutathione S-transferase (GST) is a 26 kDa protein. It has been utilized for single-step purification of fusion proteins. Due to the specific bind to glutathione coupled to a sepharose matrix, the interested enzymes fused with GST moiety retained on column matrices during the purification process. Under mild, non-denaturing conditions, the target enzymes can be released by the addition of reduced glutathione to the elution buffer. Creative Enzymes has well developed and optimized detailed protocols for successful purification of various enzymes fused with GST. Our purification process retains the solubility of the fusion protein and avoids denaturants or mild detergents during the purification.

The principle of IAC is the utilization of the high specificity between the antibody and antigen. IAC is superior to other chromatography methods when the enzyme concentration is very low in mixtures. By coupling the antibody to the appropriate resins to form an affinity adsorbent, the targeted enzymes in the fermentation matrix can be efficiently isolated and purified. Although due to the strong affinities, the elution processes may be difficult, Creative Enzymes has unique techniques to achieve efficient elution.

As the indispensable tools to achieve protein purification, affinity chromatography is the most widely applied method for biology research. Creative Enzymes provides reliable purification services. The purified enzymes are of high purity and stability. Our goal is to achieving the best quality without increasing the service cost. Please contact us for details and more advantages of our service.

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