Proteases can be used to generate small, active fragments or domains of the target protein for crystallization.⁹ The fragment or domain can be used directly for crystallization experiments. Or the proteolytic sample analyzed by gel electrophoresis and/or mass spectrometry for mass and sequence for subsequent cloning, expression, purification and crystallization. Using proteolysis to enhance sample crystallization, the current overall success rate for yielding a deposited crystal structure is currently better than 12%.³

Proti-Ace (HR2-429) contains three aliquots of 6 unique proteases (a-Chymotrypsin, Trypsin, Elastase, Papain, Subtilisin and Endoproteinase Glu-C) and six aliquots of Proti-Ace Dilution Buffer. Each protease is supplied in a stable, lyophilized format in an optimized digest buffer. Simply add water when ready to use.

Proti-Ace 2 (HR2-432) contains three aliquots of 6 unique proteases (Proteinase K, Clostripain (Endoproteinase-Arg-C), Pepsin, Thermolysin, Bromelain and Actinase E) and six aliquots of Proti-Ace Dilution Buffer. Each protease is supplied in a stable, lyophilized format in an optimized digest buffer. Simply add water when ready to use.

The unique freeze dried formulation of the Proti-Ace kits offers a much improved protease stability compared to liquid protease formulations.

Proti-Ace™

Proti-Ace™ 2

1. Allan D′Arcy, personal communication, 1989-2009.

2. In situ proteolysis for protein crystallization and structure determination. Dong, A et al. Nature Methods – 4, 1019 – 1021 (2007).

3. In Situ Proteolysis to Generate Crystals for Structure Determination: An Update. Amy Wernimont, Aled Edwards. PLoS ONE 4(4): e5094. doi:10.1371/ journal.pone.0005094.

4. The use of in situ proteolysis in the crystallization of murine CstF-77. Tong et al. Acta Cryst. (2007). F63, 135-138.

5. A brief history of protein crystal growth. McPherson, A. Journal of Crystal Growth, vol. 110, issue 1-2, pp. 1-10, 1991.

6. Preparation and analysis of protein crystals. McPherson, A. John Wiley & Sons, Inc. 1982. ISBN 089464355X.

7. A crystallizable form of the Streptococcus gordonii surface antigen SspB C-domain obtained by limited proteolysis. Forsgren et al. Acta Cryst. (2009). F65, 712-714.

8. Preliminary X-ray analysis of a human VH fragment at 1.8 angstrom resolution. Gaur, Kupper, Fischer & Hoffman. Acta Cryst. (2004). D60, 965-967.

9. Replication Protein A Characterization and Crystallization of the DNA Binding Domain. Pfuetzner et al. The Journal of Biological Chemistry, Vol. 272, No. 1, Issue of January 3, pp. 430-434, 1997.

10. Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB. Little et al. Acta Cryst. (2012) F68, 842-845.

11. Proteolysis of Native Proteins – Trapping of a Reaction Intermediate. Chenyi Wu, Duncan H. L. Robertson, Simon J. Hubbard, Simon J. Gaskell and Robert J. Beynon. doi: 10.1074/jbc.274.2.1108 January 8, 1999 The Journal of Biological Chemistry, 274, 1108-1115.

12. Crystallization of mouse RIG-I ATPase domain: in situ proteolysis. Civril F, Hopfner KP. Methods Mol Biol. 2014;1169:27-35. doi: 10.1007/978-1-4939-0882-0_3.

13. Crystallization and preliminary X-ray crystallographic analysis of YfcM: an important factor for EF-P hydroxylation. K. Kobayashi, T. Suzuki, N. Dohmae, R. Ishitani and O. Nureki. Acta Cryst. (2014). F70, 1236-1239 [ doi:10.1107/S2053230X14015726 ]. Synopsis: in situ proteolysis crystallization method produces crystals diffracting to 1.45 Å

Hampton Research, first in crystallization since 1991, developing and delivering crystallization and optimization screens, reagents, plates, and other tools for the crystallization of biological macromolecules, including proteins (antibody), peptides (insulin), and nucleic acids (DNA).

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Reductive alkylation of lysine residues to change protein properties (pI, solubility and hydropathy) which may promote crystallization via improved crystal packing.

Reductive alkylation of proteins has been successfully applied to obtain a significant number of high-quality crystals from proteins previously unable to be crystallized. Alkylating the e amino group of lysines alters the hydropathy, solubility and pI of the protein which may promote crystallization by altering sample-sample, sample-solvent and crystal packing interactions.

Reductive alkylation does not change the intrinsic charge on a protein but may change the isoelectric point (pI) slightly. The N-terminal amino group on the backbone will also be reductively alkylated. In general, alkylated proteins retain their original biochemical function. This protocol is designed with the goal of generating a high degree of modification with few side reactions, resulting in a homogeneous population of protein.

Reductive Alkylation Kit

1. Large-scale evaluation of protein reductive methylation for improving protein crystallization. Kim et al. Nature Methods, Volume 5, Number 10, page 853-854, October 2008.

2. Lysine methylation as a routine rescue strategy for protein crystallization. Walter et al. Structure 14, 1617-1622, November 2006.

3. A pivotal role for reductive methylation in the de novo crystallization of a ternary complex composed of Yersinia pestis virulence factors YopN, SycN and YscB. Florian D. Schubot and David S.Waugh. Acta Cryst. (2004). D60, 1981-1986.

4. Reductive alkylation of lysine residues to alter crystallization properties of proteins. Ivan Rayment. Methods in Enzymology, Volume 276, 171-179, (1997).

5. Crystallization and improvement of crystal quality for X-ray diffraction of maltooligosyl trehalose synthase by reductive methylation of lysine residues. Matsuura et al. Acta Cryst. (1999). D55, 931-933.

6. Structural consequences of reductive methylation of lysine residues in hen egg white lysozyme: An X-ray analysis at 1.8 angstrom resolution. Rypniewski, W.R., Holden, H.M., and Rayment, I. (1993). Biochemistry 32, 9851-9858.

7. Reductive alkylation of amino groups in proteins. Means, G. E. & Feeney, R. E. (1968). Biochemistry, 7, 2192-2201.

8. Double chromodomains cooperate to recognize the methylated histone H3 tail. Flanagan et al. Nature, 438, 1181-1185 (2005).

9. Reductive Isopropylation of Amino Groups in Lysine Containing Peptides. Brown and Greenberg. Analytical Letters, Volume 17, Issue 12 1984 , pages 1429-1445.

10. Selectivity in the Modification of the a-Amino Groups of Hemoglobin on Reductive Alkylation with Aliphatic Carbonyl Compounds. Acharya et al. Journal of Biological Chemistry, Vol. 260, No. 10, Issue of May 25, pp. 6039-6046, 1985.

11. Accessibility and mobility of lysine residues in beta-lactoglobulin. Brown et al. Biochemistry, 1988, 5601-5610.

Hampton Research, first in crystallization since 1991, developing and delivering crystallization and optimization screens, reagents, plates, and other tools for the crystallization of biological macromolecules, including proteins (antibody), peptides (insulin), and nucleic acids (DNA).

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Why use the Silica Hydrogel for crystallization? Depending upon the macromolecule and selected conditions, gels can reduce nucleation and sedimentation, provide added stability, and allow crystals to grow larger. The porous network minimizes natural convection. Crystals are suspended in the gel network so that they do not form sediments and can grow free from strain exerted by the container or other crystals. The gel fissures around a growing crystal forming a cusp-like cavity. Heterogeneous and secondary nucleation are reduced in the presence of a silica hydrogel.³ The Silica Hydrogel can be used for liquid-gel, liquid-gel-liquid, vapor diffusion, as well as dialysis crystallization methodologies. The gel is compatible with a wide range of salts, polymers, organic solvents, and buffers used for macromolecular crystallization in a pH range from 3 to 10.

Each Silica Hydrogel kit contains twelve 500 microliter tubes of sodium silicate solution and twelve 500 microliter tubes of acetic acid solution. All solutions are formulated using ultra-pure water and are sterile filtered. Crystallization accessories are sold separately.

Silica Hydrogel kit

1. Robert, M.C. & Lefaucheux, F., J. Crystal Growth (1988) 90, 358.

2. Provost, K. & Robert, M.C., J. Crystal Growth (1991) 110, 258.

3. M.C. Robert, K. Provost, & F. Lefaucheux, Crystallization of Nucleic Acids and Protein, A Practical Approach, Oxford Univ Press (1992) 127-143.

4. McPherson A., Methods in Enzymology (1985) 114, 112.

5. Cudney, B., Patel, S., McPherson, A., Acta Cryst. (1994) D50, 479-483

Hampton Research, first in crystallization since 1991, developing and delivering crystallization and optimization screens, reagents, plates, and other tools for the crystallization of biological macromolecules, including proteins (antibody), peptides (insulin), and nucleic acids (DNA).

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Agarose is a neutral polysaccharide extracted from the cellular walls of Rhodophyceae algae belonging to the genera Gelidium, Gelidiella, Pterocladia, Gracilaria, and Ahnfeltia, also known as agarophyte seaweed. The structure of the polysaccharide is that of a galactan, formed by linking agarobioses by links 1-3, 1-4. This chemical structure gives agaroses the capacity to form strong gels even at low temperatures. The gels have a macroreticular structure with a very open mesh which can be adjusted simply by varying the concentration of the agarose. The macroreticule structure of the agarose gel is formed by hydrogen bonds, which makes the gel reversible, transforming the gel into a solution by heating. The hysteresis (difference between gelling and melting temperature) is greater than any other hydrocolloid. The absence of ionic groups makes the gel a neutral structure. With no interaction, macromolecules can migrate through the gel mesh, making the gel an efficient sieve for biological macromolecules.

The LM Agarose offered by Hampton Research is 100% pure, does not contain any additives, does not contain ligation inhibitors, and is free of DNAses and RNAses. Hampton Research LM agarose is clearer than other agaroses and also has a higher gel strength.

LM AgaroseTM

1. Robert, M. C. and Lefaucheux, F. Crystal growth in gels: principle and application. J. Cryst. Growth. 90:358-367, 1988.

2. Lorber et al, Journal of Crystal Growth 204 (1999) 357-368.

3. Lorber et al, Acta Crystallographic D55 (1999) 1491-1494.

4. Provost K., Robert, M.C., Application of gel growth to hanging drop technique. Journal of Crystal Growth 110 (1991) 258-264.

5. Robert, M.C., Vidal, O., Garcia-Ruiz, J.M., and Otalora, F., Crystallization in gels and related methods. Crystallization of Nucleic Acids and Proteins, (1999)149-175, Oxford University Press, ISBN 0-

6. J.M.Garcia-Ruiz, A. Hernandez-Hernandez, J. Lopez-Jaramillo, and B. Thomas. “Crystallization screening directly in electrophoresis gels”. Journal of Crystal Growth 232, 596-602, (2001).

7. J.A. Gavira, J.M. Garcia-Ruiz. Agarose as crystallisation media for proteins II: Trapping of gel fibres into the crystals. Acta Crystallographica D 58, 1653-1656, (2002).

8. Agarose gels and the Granada Crystallization Box (http://lec.ugr.es/)

9. Basel Box, A. D′Arcy et al (2003) (method employing pipet tips and cuvettes for Counter-Diffusion crystallization).

10. Chayen, N.E., “A Novel Technique to Control the Rate of Vapour Diffusion, Giving Larger Protein Crystals” J. Appl. Cryst. 30 (1997), 198-202.

11. Chayen, N.E., “The Role of Oil in Macromolecular Crystallisation” Structure 5 (1997), 1269-1274.

Hampton Research, first in crystallization since 1991, developing and delivering crystallization and optimization screens, reagents, plates, and other tools for the crystallization of biological macromolecules, including proteins (antibody), peptides (insulin), and nucleic acids (DNA).

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