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GingisREX™ – Arginine Specific Protease

GingisREX (Rgp) protease digests proteins C-terminal to arginine residues. This enzyme can be used for analyzing proteins in mass spectrometry for peptide mapping, protein fingerprinting and sequence analysis.

GingisREX specifically digests peptides and proteins C-terminal to arginine residues. The protease is specific for Arg-X motifs and does not have activity at lysines as commonly observed using Arg-C. The enzymatic activity of GingisREX includes digestion of Arg-Pro linkages, which is difficult to digest with other enzymes. The enzyme is active at pH ranging from pH 5.0-9.0 and in presence of denaturing agents such as 6M urea and 0.1% SDS.

GingisREX™ enzyme characteristics

  • Specific activity on Arg-X residues
  • Superior specificity compared to Arg-C
  • Active on Arg-Pro linkages
  • Active in 6M urea and at pH 5.0-9.0

Applications of GingisREX™

  • Peptide mapping
  • Protein sequence analysis
  • Protein fingerprinting
  • Post-translational modification analysis

GingisREX™ has superior specificity for arginine residues compared to Arg-C

As a model substrate for GingisREX, oxidized β-chain of insulin was used to compare the activities of GingisREX and Arg-C. The digested peptides were analyzed on RP-HPLC and mass spectrometry and the data confirmed the activity at arginine residues of GingisREX and digestion at lysines of Arg-C (Figure 1, Table 1).

IOB_stack

Figure 1. Digestion of oxidized β-chain of insulin with GingisREX and Arg-C was performed O/N at 37°C, enzyme to substrate ratio 1:20 (w/w), 20mM cysteine in buffers at pH 7.4 (GingisREX) and pH 7.6 (Arg-C). The peptides were separated on RP-HPLC. Peak masses are presented in Table 1.

Peak No. Amino acid sequence Expected monoisotopic mass (Da) Measured monoisotopic mass (Da)
1 GFFYTPKA 929.5 929.5
2 FVNQHLCGSHLVEALYLVCGER 2586.4 2586.5
3 GFFYTPK 858.5 858.4
4 FVNQHLCGSH 1190.6 1190.6
5 LVEALYLVCGER + Na 1434.8 1434.5
6 FVNQHLCGSHLVEALYLVCGERGFFYTPKA 3497.9 3496.8

Table 1. Peak masses of oxidized β-chain of insulin digested by GingisREX or Arg-C, as indicated in Figure 1. Peptide number 6 refers to the intact oxidized β-chain of insulin.

GingisREX™ applied in peptide mapping of RNaseA and trastuzumab (Herceptin®)

Biopharmaceuticals are characterized throughout development and production to determine the key quality attributes such as primary sequence, oxidation, deamidation and glycosylation profile. The protease GingisREX generates larger peptides with more charge per peptide, which is advantageous for analysis by liquid chromatography and mass spectrometry. Using this workflow, the mass over charge of longer peptides can be resolved, resulting in increased sequence coverage and identification of particular PTMs.

The digested peptides of RNAseA using GingisREX or Arg-C were analyzed using LC-MS and are compared in Table 2.

On a large and complex sample, such as a therapeutic antibody, digestion at arginine residues gives larger peptides and increased sequence coverage. Additionally, having larger peptides result in fewer peaks and less complicated peptide map, which is beneficial for data interpretation in mass fingerprint analysis. As an example, the digestion profiles of trastuzumab (Herceptin®) by GingisREX and Arg-C are presented in Figure 2.

Amino acid GingisREX™ Peptide sequence Amino acid Arg-C Peptide sequence
1-10 KETAAAKFER 1-10 KETAAAKFER
11-33 QHMDSSTSAASSSNYCNQMMKSR 11-31 QHMDSSTSAASSSNYCNQMMK
11-33 QHMDSSTSAASSSNYCNQMMKSR
34-39 NLTKDR 34-39 NLTKDR
40-85 CKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCR 40-66 CKPVNTFVHESLADVQAVCSQKNVACK
40-85 CKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCR
62-85 NVACKNGQTNCYQSYSTMSITDCR
65-85 CKNGQTNCYQSYSTMSITDCR
67-85 NGQTNCYQSYSTMSITDCR
86-124 ETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV 86-104 ETGSSKYPNCAYKTTQANK
92-104 YPNCAYKTTQANK
105-124 HIIVACEGNPYVPVHFDASV

Table 2. Peptides from RNaseA digested with GingisREX or Arg-C. RNAseA was incubated with GingisREX and Arg-C at 1:50 (w/w) enzyme:protein ratio O/N at 37°C. The reactions were quenched, the peptides alkylated, separated on RP-HPLC and analyzed using MS. Detected peptide sequences are presented with arginines (R) highlighted in green and lysines (K) in red.
trasuzumab_edit
Figure 2. Peptide map of trastuzumab (Herceptin®) after digestion with GingisREX or Arg-C. Trastuzumab was incubated with GingisREX or Arg-C at 1:50 (w/w) enzyme:protein ratio O/N at 37°C at pH . The reactions were quenched, the peptides alkylated and separated on RP-HPLC.

GingisREX™ is robust and active in urea and at a broad pH range

The robustness and tolerance of GingisREX to chaotropes (urea and SDS) and detergents (Tween and SDC) were evaluated with liquid chromatography and substrate assay. The GingisREX protease tolerates high denaturing conditions and is active in 6M urea and displayed remained activity in the presence of all the tested agents although at a slower rate (Table 3). The enzyme was active at pH ranging from pH 5.0-9.0 with optimum between pH 6.5-8.0.

Chaotropic Agents and Detergents Concentration GingisREX™ activity
Urea 6M 95%
SDS 0.1%
1%
75%
50%
Tween 0.1%
1%
95%
90%
SDC 0.1%
1%
80%
70%

Table 3. GingisREX activity in chaotropic agents and detergents. The GingisREX activity was determined by enzymatic hydrolysis of a synthetic peptide substrate, 1mM L-BAPNA (Sigma) in the presence of the detergent or chaotrope in 100mM Tris-HCl, 5mM cysteine, 75mM NaCl, 2.5mM CaCl₂, pH 7.6.

Product

GingisREX is a cysteine protease that specifically digests peptide bonds C-terminal to arginine residues, including arginine linked to proline. Cysteine is required for activity and specificity of GingisREX enzyme.

Storage

The enzyme is provided as a lyophilized powder. After reconstitution by addition of water GingisREX is stable for at least 1 month at 4-8°C. The product is shipped on ice and should be stored at -20°C upon arrival.

Buffers and Reducing agent

To maintain the enzymatically active cysteine in a reduced form, TCEP is added to preserve the reducing environment (for details please consult the user instructions). GingisREX is active in a broad pH range, 5.0-9.0, with optimal activity between pH 6.5-8.0. Digestion can be performed in the presence of chaotropic agents or detergents such as urea, SDS, tween, SDC (Table 3). Buffers tested and compatible with GingisREX activity are Tris, Bis-Tris and ammonium bicarbonate.

Documents

Instructions

Product specifications

MSDS

Patent and Disclaimer

GingisREX™

The trademark GingisREX is the property of Genovis AB.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.

Methods

Digestion of insulin oxidized β-chain with GingisREX and Arg-C was performed O/N at 37°C, with enzyme to substrate ratio 1:20, in 20mM cysteine in buffers at pH 7.4 (GingisREX) pH 7.6 (Arg-C). The samples were analyzed on a Poroshell 120 EC-C18 2.7um 4.6x50mm column and a 1290 UPLC system from Agilent Technologies. The gradient was 3-60% acetonitrile in 0.1% TFA. Trastuzumab and RNaseA were digested with GingisREX and Arg-C in 4M urea, 10mM cysteine, 5mM TCEP at an enzyme:protein ratio of 1:50. Arg-C specific buffer 50mM Tris 2mM EDTA and 5mM CaCl2 pH 7.6, GingisREX 0.1M Tris pH 7.4. Digestion was performed O/N at 37°C. The reaction was quenched as the free thiols are alkylated with iodoacetamide. The peptides were separated on a slow acetonitrile gradient on an Advance BioPeptide column and a 1290 UPLC system, both from Agilent Technologies. MS data was obtained at a Thermo Scientific LTQ XL linear ion trap connected to an Accela pump and autosampler. The column was an Acquity UPLC BEH C18 1.7um 2.1x30mm in a similar gradient as above but in 0.1% FA. Insulin oxidized β-chain was obtained from Sigma-Aldrich and Arg-C from Promega Corporation.