SlyD is a putative folding helper protein from the Escherichia coli cytosol, which consists of an N- terminal prolyl isomerase domain of the FKBP type and a presumably unstructured C-terminal tail. It is involved in the biosynthesis of the metal cluster in the [NiFe]-hydrogenase enzymes, and exhibits several activities including that of a peptidyl-prolyl isomerase. Source: Recombinant protein corresponding to aa1-196 from E. coli SlyD expressed in E. coli. Molecular Weight: ~21kD (196aa) Specific Activity: ~220 nmoles/min/mg, and is defined as the amount of enzyme that cleaves 1umole of suc-AAFP-pNA/minute at 25C in Tris-HCL, pH 8.0, using chymotrypsin. Activity Assay: 1. Prepare 170ul assay buffer into a suitable container and pre-chill on ice before use: The final concentrations are 200mM Tris-HCL, pH 8.0, and 20nM chymotrypsin. 2. Add 10ul of recombinant SlyD protein with 1ug in assay buffer. 3. Mix by inversion and equilibrate to 1C and monitor the A405nm until the value is constant using a spectrophotometer. 4. Add 20ul pre-chilled 5mM suc-AAFP-pNA. (Substrate was dissolved in TFE that contained 460mM LiCl to a concentration of 3mM) 5. Record the increase in A405 nm for 30 minutes at 25C. Amino Acid Sequence: MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDV AVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVD GNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDGCCGG HGHDHGHEHG GEGCCGGKGN GGCGCH Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
