PC-5 (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-PC5
In stock
- SKU
- REC-PC5
Target Overview
PC-5 (Recombinant) is a recombinant form of human proprotein convertase subtilisin/kexin type 5 (PCSK5; UniProt Q92824), produced by transient expression in HEK293 cells. PCSK5 encodes a serine endoprotease of 1,860 amino acids belonging to the subtilisin/kexin family of proprotein convertases. HEK293 expression is the preferred system for this target because it supports the mammalian-type glycosylation and signal peptide processing characteristic of the native secreted enzyme, improving the likelihood of correct folding and catalytic activity relative to prokaryotic platforms. The enzyme cleaves substrate proproteins at paired basic residue sites conforming to the RXXX[KR]R consensus motif, operating in both constitutive and regulated secretory pathways. Confirmed substrates include bone morphogenetic protein 2 (BMP2), caldesmon (CALD1), and α-integrins, placing PCSK5 at an intersection of extracellular matrix remodelling, growth factor activation, and cell adhesion signalling. In the laboratory, this recombinant is used primarily in three contexts. First, endoprotease activity assays: researchers measure cleavage of fluorogenic or peptide substrates bearing the RXXX[KR]R motif to characterise enzymatic kinetics (Km, kcat) or screen small-molecule inhibitor libraries for IC50 determination. Second, substrate mapping: the recombinant can be incubated with candidate proprotein substrates to confirm cleavage site identity by mass spectrometry or gel-based readouts. Third, antibody validation: researchers working with PCSK5-directed antibodies — including the matched Triple Point Biologics antibody (RP-PC5) — use this recombinant as a positive control antigen in Western blot titration and immunoprecipitation experiments, confirming specificity against a well-defined, species-matched standard.
Background
Applications
- Fluorogenic peptide substrate cleavage assay to measure PCSK5 endoprotease kinetics (Km, kcat) using RXXX[KR]R-motif substrates
- Small-molecule inhibitor IC50 determination in a proprotein convertase selectivity panel
- Substrate identification and cleavage-site mapping by incubation with candidate proproteins followed by LC-MS/MS analysis
- Positive control antigen for Western blot validation of anti-PCSK5 antibodies, including matched antibody RP-PC5
- Immunoprecipitation input control to confirm antibody specificity in pull-down experiments
- ELISA capture or detection standard for PCSK5 quantification assay development
- Biophysical characterisation (SPR, ITC) of PCSK5 interactions with inhibitors or substrate peptides
- In vitro proprotein processing assay using recombinant BMP2 or integrin precursor substrates to confirm PCSK5-dependent activation
References
Additional Specifications
| Storage Buffer | 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol |
|---|---|
| Endotoxin Level | <0.1 EU/µg by LAL |
| Purity (%) | >90% by SDS-PAGE |
| Expression System | HEK293 |
| Subcellular Localization | Subcellular localization not yet annotated |
Frequently Asked Questions
What is the expected molecular weight of recombinant PC-5 on SDS-PAGE or Western blot?
PCSK5 (PC-5) is a 1,860 amino acid protein with a predicted unglycosylated MW of approximately 207 kDa. Because this recombinant is produced in HEK293 cells, mammalian N-linked glycosylation adds significant mass; expect the apparent MW on SDS-PAGE and Western blot to run between 230–270 kDa, depending on gel percentage and running conditions. A 6–8% acrylamide gel is recommended for adequate resolution in this size range. The HEK293 expression system also supports correct signal peptide cleavage, so the predominant band represents the processed, secreted form.
Which isoform or processing form of PCSK5 does this recombinant correspond to?
PC-5 (Recombinant) corresponds to the secreted isoform of human PCSK5 (UniProt Q92824), with signal peptide processing occurring during production in HEK293 cells. PCSK5 is synthesized as a zymogen that undergoes autocatalytic removal of its N-terminal prodomain in the endoplasmic reticulum before export. The active enzyme form delivered in this vial retains the catalytic, P-domain, and cysteine-rich C-terminal region. If your experiment requires the full-length unprocessed zymogen as a reference, this product is not the appropriate control.
What substrates does recombinant PC-5 cleave and what consensus motif should I design my assay around?
PCSK5 cleaves proproteins at paired basic residue sites conforming to the RXXX[KR]R↓ consensus motif. Confirmed endogenous substrates include BMP2 (pro-domain cleavage to release active growth factor), caldesmon (CALD1), and α-integrin precursors. For fluorogenic activity assays, peptides based on the BMP2 furin-cleavage site (e.g., Ac-RQKR-AMC or optimized PCSK-family substrates containing the RXXX[KR]R motif) are widely used. Pyr-RTKR-AMC is a validated generic PC-family substrate and provides a reliable starting point for kinetic characterization of this recombinant.
What buffer and assay conditions work best for a PC-5 protease activity assay?
For fluorogenic peptide cleavage assays, we recommend diluting PC-5 (Recombinant) into a reaction buffer of 100 mM HEPES pH 7.0–7.4, 1 mM CaCl₂, and 1 mM β-mercaptoethanol (or 0.5 mM DTT). The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible at low carry-over volumes. PCSK family enzymes require calcium for activity; do not use EDTA-containing buffers. Optimal temperature is 37°C. A typical starting enzyme concentration is 5–20 nM; titrate against your substrate to establish linear signal kinetics before running inhibitor or activation experiments.
What starting concentration of recombinant PC-5 should I use in an initial activity assay?
Start at 10 nM PC-5 (Recombinant) with 50–100 µM of a fluorogenic substrate (e.g., Pyr-RTKR-AMC) in a 100 µL reaction volume at 37°C. Monitor AMC release at Ex/Em 380/460 nm over 30–60 minutes. If the signal is low or the linear range is narrow, titrate enzyme from 5 to 50 nM. Because activity can vary with freeze-thaw history, include an enzyme-only no-substrate blank and a substrate-only no-enzyme blank in every plate. Single-use aliquots are provided precisely to avoid the activity loss associated with repeated freeze-thaw cycling.
Can I use PC-5 (Recombinant) as a positive control for Western blot with the matched RP-PC5 antibody?
Yes. PC-5 (Recombinant) (REC-PC5) is the designated positive control for the matched rabbit polyclonal antibody RP-PC5 (see /anti-pc-5-rabbit-polyclonal-antibody). Both products are produced and validated in the same laboratory, so epitope compatibility is confirmed rather than assumed. RP-PC5 detects denatured PCSK5 under standard reducing SDS-PAGE conditions. Load 50–100 ng of recombinant protein per lane on a 6–8% gel for a clean, unambiguous band at ~230–270 kDa. This pairing is particularly useful when validating RP-PC5 in a new cell or tissue lysate where endogenous PCSK5 expression is uncertain.
How much PC-5 (Recombinant) should I load as a Western blot positive control alongside my cell lysate?
50–100 ng per lane is the recommended loading range when using RP-PC5 as the primary antibody. At 50 ng you should see a distinct band with low background; at 100 ng the signal is robust enough to serve as a reliable molecular weight marker lane for PCSK5. Run the recombinant in a lane adjacent to your lysate sample so band alignment is direct. If you observe signal at an unexpected MW in your lysate, the recombinant lane confirms the antibody is detecting the correct target at the predicted size, ruling out cross-reactivity as the source of the aberrant band.
How should I store and handle PC-5 (Recombinant) to maintain activity, and what is the shelf life after thawing?
PC-5 (Recombinant) is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at −20°C in the single-use aliquots provided. Avoid repeated freeze-thaw cycles, which progressively reduce catalytic activity. Once thawed, keep the tube on ice and use within the same working session; do not refreeze. For working dilutions, use your assay buffer (not PBS alone, which lacks the glycerol stabilizer) and prepare fresh. At −20°C in the original aliquots, expected shelf life is 12 months from the date of receipt; this assumes the cold chain was maintained during shipping.
Validation imagery coming soon
Western blot validation figures for REC-PC5 will be published here as they are produced in-house.
If you would like to see existing validation data for this antibody before publication, request a sample copy.