Protease
Recombinant Protease
Cat. No.
BT4103
Source
Escherichia Coli.
Synonyms
Appearance
Sterile filtered colorless solution.
Purity
Usage
Prospec's products are furnished for LABORATORY RESEARCH USE ONLY. They may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.
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Description
Protease Recombinant is a fusion protein of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease. The protease specifically recognizes a subset of sequences which include the core amino acid sequence Leu-Phe-Gln/Gly-Pro cleaving between the Gln and Gly residues. Substrate recognition and cleavage are likely to be dependent not only upon primary structural signals, but also upon the secondary and tertiary structures of the fusion protein as well.
The Recombinant Protease is purified by proprietary chromatographic techniques.
The Recombinant Protease is purified by proprietary chromatographic techniques.
Product Specs
Description
Protease Recombinant is a fusion protein composed of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease. Its primary function is to recognize and cleave a specific set of sequences containing the core amino acid sequence Leu-Phe-Gln/Gly-Pro, specifically targeting the bond between Gln and Gly residues. It's important to note that substrate recognition and cleavage might be influenced by the fusion protein's primary, secondary, and tertiary structures. The purification process of this recombinant protease involves proprietary chromatographic techniques.
Physical Appearance
A clear, colorless solution that has been sterilized by filtration.
Cleavage Buffer
The recommended cleavage buffer consists of 50mM Tris-HCl at a pH of 7.0 (measured at 25 degrees Celsius), 150mM NaCl, 1mM EDTA, and 1mM dithiothreitol. Ensure that the buffer is chilled to 5 degrees Celsius before use.
Cleavage Conditions
When performing cleavage reactions with a fusion protein, it is advisable to collect samples at different time intervals and analyze them via SDS-PAGE. This helps in assessing the yield, purity, and the progress of digestion. The optimal amount of PreScission Protease, incubation temperature, and duration required for complete digestion of a GST fusion partner can vary depending on the specific fusion partner. Therefore, it's crucial to determine the optimal conditions for each fusion through preliminary experiments. Adding Triton X-100, Tween 20, Nonidet, or NP40 to a final concentration of 0.01% may enhance digestion. Note that concentrations of these detergents up to 1% do not inhibit PreScission Protease.
Stability
For optimal storage, keep the protease refrigerated at 4 degrees Celsius if you plan to use the entire vial within 2 to 4 weeks. If longer storage is needed, freeze the protease at -20 degrees Celsius. Minimize the number of times you freeze and thaw the protease.
Unit Definition
One unit of this protease is defined as the amount required to cleave approximately 90% of a 100 microgram sample of a test GST-fusion protein. This cleavage reaction is carried out in a specific cleavage buffer (containing 50mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, at a pH of 7.0 and a temperature of 25 degrees Celsius) and is incubated at 5 degrees Celsius for 16 hours.
Source
Escherichia Coli.
Product Science Overview
Introduction
Recombinant proteases are enzymes that catalyze the hydrolysis of peptide bonds in proteins. These enzymes are produced through recombinant DNA technology, which involves inserting the gene encoding the protease into a host organism to produce the enzyme in large quantities. Recombinant proteases have significant applications in various industries, including biotechnology, pharmaceuticals, and food processing.
Recombinant DNA Technology
Recombinant DNA technology is the cornerstone of producing recombinant proteases. This technology involves combining DNA from different sources to create a new genetic sequence. The process typically includes the following steps:
- Isolation of the Gene: The gene encoding the desired protease is isolated from the source organism.
- Insertion into a Vector: The isolated gene is inserted into a plasmid or another type of vector, which can replicate within a host cell.
- Transformation: The vector containing the recombinant DNA is introduced into a host cell, such as bacteria, yeast, or mammalian cells.
- Expression: The host cells are cultured under conditions that promote the expression of the recombinant protease.
- Purification: The expressed protease is extracted and purified for use in various applications.
Expression Systems
The choice of expression system is crucial for the successful production of recombinant proteases. The most commonly used systems include:
- Prokaryotic Systems: Bacteria, such as Escherichia coli and Bacillus species, are frequently used due to their rapid growth and ease of genetic manipulation. However, they may not always produce proteases with the correct post-translational modifications.
- Eukaryotic Systems: Yeast, insect, and mammalian cells are used when post-translational modifications are essential for the protease’s activity. These systems can produce more complex proteins but are generally more challenging to work with and require more stringent culture conditions .
Applications
Recombinant proteases have a wide range of applications:
- Biotechnology: They are used in protein engineering, structural biology, and the production of recombinant proteins.
- Pharmaceuticals: Recombinant proteases are used in drug development and as therapeutic agents. For example, they can be used to produce insulin and other peptide-based drugs.
- Food Processing: Proteases are used in the dairy industry for cheese production, in the brewing industry for beer clarification, and in meat tenderization .
Advances and Challenges
Recent advancements in recombinant protease production include the use of rational design and directed evolution to enhance enzyme activity and stability. These techniques allow for the creation of proteases with improved properties tailored to specific industrial applications. However, challenges remain, such as optimizing expression systems for high yield and ensuring the correct folding and activity of the recombinant proteases .
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