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JBS Floppy-Choppy

In situ proteolysis as rescue technique in protein crystallization

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CO-110 1 Kit 178,00 Add to Basket/Quote Add to Notepad

For in vitro use only!

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Applications:
In situ proteolysis of protein samples by different proteases in the crystallization trial to enhance crystallization behavior of the protein.

Description:
Analysis of structural genomics surveys show that only 15-20 % of the protein targets which can be purified will yield single crystals suitable for X-ray structure determination [1,2].

Beside the possibility to screen and optimize crystallization conditions, the protein itself can be modified to enhance the crystallization behavior. A prominent example is the reductive methylation of lysine residues on the protein surface (see JBS Methylation Kit, CO-510).

Further, it has been shown that domains or stable fragments of proteins crystallize a lot easier or yield better diffracting crystals than the intact protein. The addition of trace amounts of proteases to the protein solution immediately prior to crystallization - in situ proteolysis - generally results in digestion of flexible parts of the protein, i.e. trimming of the N- and/or C-termini as well as common 'tags' (such as His6). In a few instances internal loop digestion and entire domain removal has been observed [2].

Large scale application has shown that in situ proteolysis has doubled the success rate in protein crystallization and structure determination and therefore is one of the most efficacious crystallization rescue strategies [2].

Content:
5 aliquots of 50 μl stock solutions, each of:

  • 1 mg/ml α-chymotrypsin (supplied in 1 mM HCl and 2 mM CaCl2)
  • 1 mg/ml Trypsin (supplied in 1 mM HCl and 2 mM CaCl2)
  • 1 mg/ml Subtilisin A (supplied in 10 mM Na acetate and 5 mM Ca acetate)
  • 1 mg/ml Papain (supplied in water)
4 aliquots of 1 ml buffer, containing 10 mM HEPES pH 7.5 and 500 mM NaCl

JBS Floppy-Choppy for in situ proteolysis

  • α-Chymotrypsin from bovine pancreas
    EC- Number: 3.4.21.1
    α-Chymotrypsin is a serine protease that selectively catalyzes the hydrolysis of peptide bonds at the carboxyl side of tyrosine, tryptophan and phenylalanine.
  • Trypsin from bovine pancreas
    EC- Number: 3.4.21.4
    Trypsin belongs to the serine protease S1 family. It cleaves peptides at the carboxyl side of arginine and lysine except when either is followed by proline. Acidic residues on either side of the cleavage site lower the rate of hydrolysis.
  • Subtilisin from Bacillus licheniformis
    EC- Number: 3.4.21.62
    Subtilisin A is grouped into the Serine S8 Endoproteinase family. It shows a broad specificity with a preference for a large uncharged residue in the P1 position. It is active under alkaline conditions.
  • Papain from Papaya latex
    EC- Number: 3.4.22.2
    Papain is a cysteine protease belonging to the peptidase C1 family. It catalyzes the hydrolysis of peptide bonds of basic amino acids, leucine or glycine. It also hydrolyzes esters and amides.

Pre-screening to identify a promising protease

Limited proteolysis can be monitored by denaturing gel electrophoresis or mass spectrometry in order to identify a suitable protease and its appropriate

Instructions for pre-screening:

  • Prepare a 1:100 dilution of an aliquot of each protease using the supplied buffer containing 10 mM HEPES pH 7.5 and 500 mM NaCl.
  • Transfer approximately 100 μg of protein (5-10 μl @ 20-10 mg/ml) into 4 vials each.
  • Add 10 μl of diluted protease (0.1 μg) per vial and incubate for thirty minutes at room temperature.
  • Quench reaction with SDS-Coomassie sample loading buffer (for gel electrophoresis) or formic acid (for mass spectrometry).
  • Analyze by SDS-PAGE or MS and select the protease which yielded the largest, most stable digestion product for subsequent crystallization experiments.
  • In case that insufficient digestion is observed repeat steps 1-5 with a 1:10 dilution of each protease.
  • In case that overdigestion is observed, repeat steps 1-5 with a 1:1,000 dilution of each protease.

In situ proteolysis

In situ proteolysis implies the addition of trace amounts of protease to the protein solution immediately prior to crystallization. Thus, crystallization experiments can be set up without evaluating the efficacy of proteolysis, without stopping the proteolysis reaction and without purification of any proteolyzed protein fragments.
However, if the protein sample is scarce, we recommend pre-screening to identify a suitable protease and concentration as described above.

Instructions for In situ proteolysis

  • Allow the protease stock solutions to thaw on ice.
  • Add protease stock solution to the purified protein on ice immediately prior crystallization trials. Use the amount of protease identified during pre-screening.

  • li>If no pre-screening has been performed, use 1 μg of protease per 100 - 10,000 μg of protein, i.e. for a ratio of 1:100 (w/w) add 10 μl of protease stock to 100 μl of protein @ 10 mg/ml.
  • Perform crystallization experiments as usual.

Selected Literature Citations of JBS Floppy-Choppy

Ochi et al. (2012) Structural Insights into the Role of Domain Flexibility in Human DNA Ligase IV. Structure 20(7):1212

Abskharon et al. (2011) Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes. PEDS 24(9):737

Selected References:
[1] Dong et al. (2007) In situ proteolysis for protein crystallization and structure determination. Nature Meth. 4:1019.
[2] Wernimont et al. (2009) In Situ Proteolysis to Generate Crystals for Structure Determination: An Update. PLoS ONE 4:e5094.