In situ proteolysis as rescue technique in protein crystallization
For in vitro use only!
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
In situ proteolysis of protein samples by different proteases in the crystallization trial to enhance crystallization behavior of the protein.
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 .
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 .
5 aliquots of 50 μl stock solutions, each of:
JBS Floppy-Choppy for in situ proteolysis
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:
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
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
 Dong et al. (2007) In situ proteolysis for protein crystallization and structure determination. Nature Meth. 4:1019.
 Wernimont et al. (2009) In Situ Proteolysis to Generate Crystals for Structure Determination: An Update. PLoS ONE 4:e5094.