The Protein Man's Blog | A Discussion of Protein Research

 In a cell, protein degradation and synthesis maintain the balance of proteins, where old proteins are degraded into amino acids, which are further used in the synthesis of new proteins. Proteases/Peptidases play an important role in protein degradation. Proteases are present in almost all living cells. In a living cell, protease action is regulated by external as well as internal factors. Some proteases are inactive under normal conditions, but are activated only when necessary. For example, during apoptosis, Pro-caspase (inactive) converts to Caspase (active). Sub-cellular localization of proteases also helps in their regulated activity. Most of them are localized to sub-cellular organelles like lysosomes and thereby restricted their activity only to those proteins directed to lysosomes. 

During cell lysis, proteases are released into the lysate and also their regulated mechanisms are disrupted. So these proteases act on various proteins in the lysate and act on a variety of proteins in cell lysate, including the protein of interest. To prevent this undesired protein degradation, proteases should be inactivated before their action. Proteases are classified based on their preference amino acid, active site metal on the target protein, as Serine Proteases, Cysteine Proteases, Threonine Proteases, Metallo Proteases, Aminopeptidases, Trypsin and related proteins, Calpain, etc., Protease activity is inhibited by protease inhibitors. Not all proteases are inhibited by a single inhibitor, so there is a need for use of a variety of protease inhibitors to inhibit protease activity. Hence cocktail of various inhibitors is added to the buffer before cell lysis and also some proteases require the presence of inhibitor throughout the purification process to avoid protein degradation.

            Protease inhibitor cocktails are a mixture of more than two inhibitors used to inhibit various types of Proteases. Components and concentration of a protease inhibitor depend on the type of cells to be lysed, downstream processing and application. Proteases vary from cell type. Protease inhibitors are added during homogenization to inhibit the proteases in the cell lysate. Some examples,

1. AEBSF, (4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride) is a Serine Protease inhibitor. It is active in low pH buffers.
2. Bestatin (Ubenimex), is an aminopeptidase inhibitor. (Inhibits Arginyl, Alanyl, Cystinyl, Leucyl Aminopeptidase, LTA4H membrane dipeptidase).
3. E 64-(Epoxide) is a cysteine peptidase inhibitor. (Inhibits Papain, Cathepsin-B, Cathepsin L; Calpain, Staphopain).
4. Pepstatin-A, an Aspartyl protease inhibitor (Inhibits Pepsin, Cathepsin-D, Cathepsin E).
5. PMSF, (Phenyl methyl sulfonyl Fluoride) is a Serine Protease inhibitor. It is active at neutral pH. It binds to serine in the active site and inhibits the protease.
6. EDTA inhibits metalloproteases. It chelates metal ions, hence not suitable for experiments with IMAC columns.
7. Aprotinin inhibits Trypsin and related enzymes.
8. Leupeptin inhibits Cysteine, Serine, and Threonine Peptidases.
9. 1,10-Phenanthroline is a Zinc Metallopeptidase inhibitor (inhibits Carboxypeptidase-A).
10. Benzamidine is an inhibitor of Serine proteases, Trypsin, and related enzymes.
11. Iodoacetamide is an inhibitor of cysteine peptidases.
12. Calpain inhibitor inhibits Calpain, a non-lysosomal cysteine protease.
13. Phosphoramidon is a membrane-metallo endopeptidase inhibitor.

G-Biosciences offer a variety of protease inhibitor cocktails supplied as 100 X buffered solutions suitable for bacterial, mammalian, plant, fungal/yeast cell lysis, and cocktail suitable for proteomic analysis such as 2D and mass spec. During recombinant protein purification from bacterial cells, some factors are to be considered like His-tagged protein purification, the presence of EDTA affects the binding of the protein to metal affinity chromatographic column. Hence there is a need for use of EDTA-free protease inhibitors. Recom ProteaseARREST™ is a cocktail suitable for recombinant protein purification and it is free of EDTA. Mammalian secretory proteins are released into culture media, these proteins are exposed to various external factors resulting in degradation, hence there is a need to include protease inhibitors in culture media during cell growth. TCM ProteaseARREST™ can be used for these experiments.

References:

1. Gottesman, S. (1996). Proteases and their targets in Escherichia coli. Annual Review of Genetics, 30(1), 465–506.
2. Chabbat, J. et al. Aprotinin is a competitive inhibitor of the factor VIIa-tissue factor complex. Thrombosis Research, Volume 71, Issue 3, 205 - 215.
3. Eri Govrin, Alex Levine, Purification of Active Cysteine Proteases by Affinity Chromatography with Attached E-64 Inhibitor, Protein Expression and Purification, Volume 15, Issue 3, 1999, Pages 247-250.