Cystatin 4, also known as Cystatin-S, Salivary Acidic Protein 1, and Cystatin-SA-III, is a member of the cystatin superfamily. This family encompasses proteins that contain multiple cystatin-like sequences. Some members of this family are active cysteine protease inhibitors, while others have lost or possibly never attained this inhibitory activity .
Cystatin 4 is a secreted protein that is expressed in submandibular and sublingual saliva but not in parotid saliva at the protein level. It is also found in other bodily fluids such as tears, urine, and seminal fluid . The recombinant form of Cystatin 4 produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 131 amino acids (21-141 a.a.) and has a molecular mass of 15.4 kDa. Under reducing conditions, it migrates at 13.5-18 kDa on SDS-PAGE .
Cystatin 4 strongly inhibits papain (non-competitively) and ficin, partially inhibits stem bromelain and bovine cathepsin C, but does not inhibit porcine cathepsin B or clostripain . This selective inhibition is significant for understanding its role in various physiological processes and potential therapeutic applications.
The recombinant Cystatin 4 is expressed with a 10 amino acid His tag at the C-terminus and purified using proprietary chromatographic techniques . The protein solution is formulated in Phosphate Buffered Saline (pH 7.4) with 10% glycerol, and it is recommended to store it at 4°C if used within 2-4 weeks or at -20°C for longer periods. For long-term storage, adding a carrier protein (0.1% Human Serum Albumin or Bovine Serum Albumin) is advised to avoid multiple freeze-thaw cycles .
Cystatin 4 has various applications in laboratory research, particularly in studying cysteine protease inhibition and its implications in different biological processes. It is important to note that products like Cystatin 4 are intended for laboratory research use only and are not suitable for use as drugs, agricultural or pesticidal products, food additives, or household chemicals .