1LEC, IACT (CSIC-UGR). Ed. Instituto López Neyra. P.T ... · This research is a product of the...
1
Coupling counterdiffusion and micro-seeding techniques to increase protein crystallization succeed 1 LEC, IACT (CSIC-UGR). Ed. Instituto López Neyra. P.T. Campus de la Salud. 18100. Granada, Spain. 2 Douglas Instruments Ltd., East Garston, Hungerford, BERKS, RG17 7HD, UK. INTRODUCTION EXPERIMENTAL RESULTS REFERENCES This work was supported by the OptiCryst project of the VI European Framework Program and the Andalusian Regional Government, Spain, project RNM 5384. This research is a product of the “Factoría Española de Cristalización”, a project Consolider- Ingenio 2010 of the Ministerio de Innovación y Ciencia (MICINN) of Spain Hernández-Hernández Ma. Angeles 1 , González-Ramírez Luis A. 1 , F. M. Baldock Peter 2 , A. Briggs Richard 2 , A. Kolek Stefan 2 , D. Shaw Stewart Patrick 2 , Gavira Jose A. 1 * CONCLUSIONS Protein Protein concentration and seed preparation conditions Micro-seeding conditions Glucosa Isomerase 20 mg/ml, Batch experiment using 100 mM Hepes pH 7.0 and 0.9 M Ammonium sulfate. 30 mg/mL 10 mg/mL 10 mg/mL + seeds 10 mg/mL + dipped in seeds Ribonuclease-A 240 mg/mL, Counterdiffusion technique, precipitating solution C21**: 6 M Na-Formate. 240 mg/mL 50 mg/mL 50 mg/mL + seeds 50 mg/mL + dipped in seeds Catalase 36 mg/mL, Vapor diffusion technique using the crystallization mushroom. Precipitating solution 10% w/v PEG-4000. 40 mg/mL 7 mg/mL 7 mg/mL + seeds 7 mg/mL + dipped in seeds Myoglobin 80 mg/mL, Counterdiffusion technique, precipitating solution C1**: 30% PEG 4K, 0.2 M Mg Chloride, 0.1 M Tris-HCl pH 8.50 80 mg/mL 5 mg/mL 5 mg/mL + sedds 5 mg/mL + dipped in seeds α-Lactalbumin 72 mg/mL, Hanging drop technique. Precipitating solution C23**: 0.82 M K- Phosphate, 0.82 M Na-Phosphate, 0.1 M Na- Hepes pH 7.50. 30 mg/mL 5 mg/mL 5 mg/mL + sedds 5 mg/mL + dipped in seeds Thaumatin 30 mg/mL, D5 from Jena Bioscience Membrane screen 3; 1.5 M Lithium Sulfate, 100 mM HEPES Sodium Salt, pH 7.5 90 mg/mL 10 mg/mL 10 mg/mL + sedds 10 mg/mL + dipped in seeds Xylanase 36 mg/mL, No.45 from Molecular Dimensions Structure Screen1 ; 4.0M Sodium Formate 56 mg/mL 36 mg/mL 36 mg/mL + sedds 36 mg/mL + dipped in seeds Trypsin 30 mg/mL, D6 from Jena Bioscience Membrane screen 3; 2.0M Sodium Chloride, 100mM Sodium Formate 90 mg/mL 30 mg/mL 30 mg/mL + sedds 30 mg/mL + dipped in seeds Concanavalin 5 mg/mL, B8 from (Molecular Dimensions) JCSG plus screen; 10% w/v PEG 8000, 100ml Tris-HCl, pH 7.0, 200mM Magnesium Chloride 15 mg/mL 5 mg/mL 5 mg/mL + sedds 5 mg/mL + dipped in seeds Figure 1. Protein crystals microphotographs. Capital letters show the seeds used, small letter crystals after seeding. A) Glucosa Isomerase crystals obtained by batch 20 mg/mL and 100 mM Hepes pH 7.0 and 0.9 M Ammonium sulfate, a) Glucosa Isomerase crystals after seeding 10 mg/mL condition C2** B) Catalase crystallized at 36 mg/mL by vapor diffusion technique and 10% w/v PEG-4000, b) Catalase at 7 mg/mL condition C8**, C) Myoglobin obtained at 20 mg/mL condition C1**; c) Myoglobin crystallized at 5 mg/mL condition C1**; D) Thaumatin 30 mg/mL and precipitated with D5 from Jena Bioscience Membrane screen3; 1.5 M Lithium Sulfate, 100 mM HEPES Sodium Salt, pH 7.5; d) Thaumatin 30 mg/mL and obtained in condition C7**; E) α-Lactalbumin crystallized at 72 mg/mL by Hanging drop technique and 0.82 M K- Phosphate, 0.82 M Na- Phosphate, 0.1 M Na-Hepes pH 7.50.; e) α-Lactalbumin crystallized at 5 mg/mL. C13**; F) Ribonuclease-A obtained at 240 mg/mL condition C21**; f) Ribonuclease-A crystallized at 50 mg/mL condition C21**. A a B b C c D d E e F f ** Counterdiffusion Screening Kit ® ref. GCB-CSK-24. See the last part appendix the composition of the 24 conditions. TABLE 1. Crystallization conditions for initial protein micro- seeds preparation and the final condition to study micro- seeding. 1 2 3 4 0 1 2 3 4 5 6 7 8 Hits number Ribonuclease-A 240 mg/mL 50 mg/mL 50 mg/mL + seed 50 mg/mL + dipped in seeds 1 2 3 4 0 1 2 3 4 5 6 7 8 Hits number Myoglobin 80 mg/mL 5 mg/mL 5 mg/mL + seed 5 mg/mL + dipped in seeds 1 2 3 4 0 3 6 9 12 15 Hits number - Lactalbumin 30 mg/mL 5 mg/mL 5 mg/mL + seed 5 mg/mL + deeped in seeds 1 2 3 4 0 2 4 6 8 10 12 14 Hits number Tripsin 90 mg/mL 30 mg/mL 30 mg/mL + seeds 30 mg/mL + dipped in seeds 1 2 3 4 0 5 10 15 20 25 Hits number Concanavalin 15 mg/mL 5 mg/mL 5 mg/mL + seeds 5 mg/mL + dipped in seeds 1 2 3 4 0 2 4 6 8 10 12 14 10 mg/mL + dipped in seeds 10 mg/mL + seeds Hits number Glucose Isomerase 30 mg/mL 10 mg/mL 1 2 3 4 0 2 4 6 8 10 12 14 10 mg/mL + dipped in seeds 10 mg/mL + seeds Hits number Thaumatin 90 mg/ml 10 mg/mL 1 2 3 4 0 2 4 6 8 10 12 14 16 18 20 Hits number Catalase 40 mg/mL 7 mg/mL 7 mg/mL + seed 7 mg/mL + dipped in seeds 1 2 3 4 0 2 4 6 Hits number Xilanase 56 mg/mL 36 mg/mL 36 mg/mL + seeds 36 mg/mL + dipped in seeds TABLE 2. Number of hits in the protein crystallization study in capillaries by combination of counterdiffusion and micro-seeding techniques using different model proteins. NOTE: The last capillary correspond to the filled with the proteins solution and after dipped in the Seed-Syrup Stock solution also named as “touch”. For the first time, it is reported the crystallization of several model proteins using micro- seeding by the counterdiffusion technique. Overall, most of the protein studied (90%) increase the number of hits in which the nucleation occurs even when the capillary only touches the “seeds syrup” . The most effective protocol was mixing the protein solution with the seed-syrup, which produced an homogeneous distribution of seeds along the capillary. It is well known that the initial seed may influence definitively the quality of the final crystals. However in this work the use of (in appearance) not so good quality crystals, such as the case of spherulites of Myoglobine, was done on purpose resulting in the growth of faceted crystals. Seeds from the initial crystalline-precipitate can be used to set-up a minimum counter- diffusion screening experiment, even at low protein concentration, to find new crystallization conditions and to produce crystals of improved visual quality. Distinct methods have being studied to improve quality and size of protein crystals. Protein crystallization in capillaries by counterdiffusion technique has been used routinely for crystal optimization. On the other hand, seeding techniques has open a possibility to improve protein crystal quality and size by carrying on new crystallization experiments on the metastable region of the phase diagram where only growth can occur. Ideally, seeding should be implemented using batch technique to avoid subsaturation conditions that could dissolve the seed or, high supersaturation conditions as can be obtained in vapour diffusion technique. Counterdiffusion technique allows the introduction of seeds into a system where the mass transport is controlled by diffusion in which supersaturation increase slowly enough to permit the growth of several seeds minimizing cross influence, mixing and conventions effects. In this work we present the proof of this concept producing optimized crystals of several model proteins by the combination of counterdiffusion and micro-seeding techniques. • A. McPherson, in: Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press, New York, 1998 (Appendix section). • Enrico A. Stura and Ian A. Wilson, (1990). Analytical and Production Seeding Techniques. METHODS: ACompanion to Methods in Enzymology. Vol. 1, No. 1, August, pp. 38-49. • J.M. García-Ruiz, (2003). Counterdiffusion Methods for Macromolecular Crystallization. Methods in Enzymology, Vol. 368 130-154. • R.J. Read and J.L. Sussman (eds.): Evolving Methods for Macromolecular Crystallography, (Succeeding with seeding: some practical advice, Terese Berfors) 1–10 © 2007. Springer • T. Bergfors, (2003). Seeds to crystals, Journal of Structural Biology 142, 66–76. • E. Saridakis,K. Dierks, A. Moreno, M. W. M. Dieckmannd and N. E. Chayen. (2002). Separating nucleation and growth in protein crystallization using dynamic light scattering. Acta Cryst. D58, 1597-1600. ACKNOWLEDGEMENTS E-mail: [email protected] , [email protected] , ** [email protected] Seed-Syrup preparation 200 mL protein solution + Crystalline material Crystals were crushed using a glass capillary [protein] [protein] = CONTROL 1 2 Protein + Seed- Syrup Seed-Syrup Stock and capillary “touch” 3 4 Capillaries 1 2 3 4 Crystalline material Spherules, amorphous aggregates, needle or micro-crystals aggregates. Obtained by different techniques (Bath, vapour diffusion, counterdiffusion) Counterdiffusion Screening Kit ® GCB-CSK-24 **. One of the goals of this study was to increase the number of hits during the screening in capillaries by counterdiffusion using the minimum Kit (24 conditions) using micro-seeding technique. Our results show that both protocols, mixing the seed with the protein solution and touching the seed syrup, increase the number of hits when compare with the low concentration protein solution (90%). In three cases (33%) the number of hits using seeds was higher than those obtained with high protein concentration solution and in one case we got crystals only when seeds were included.
Transcript of 1LEC, IACT (CSIC-UGR). Ed. Instituto López Neyra. P.T ... · This research is a product of the...
Coupling counterdiffusion and micro-seeding techniques to increase protein crystallization succeed
1LEC, IACT (CSIC-UGR). Ed. Instituto López Neyra. P.T. Campus de la Salud. 18100. Granada, Spain. 2Douglas Instruments Ltd., East Garston, Hungerford, BERKS, RG17 7HD, UK.
INTRODUCTION EXPERIMENTAL
RESULTS
REFERENCES
This work was supported by the OptiCryst project of the VI European Framework Program and the Andalusian Regional Government,Spain, project RNM 5384. This research is a product of the “Factoría Española de Cristalización”, a project Consolider- Ingenio 2010 of theMinisterio de Innovación y Ciencia (MICINN) of Spain
Hernández-Hernández Ma. Angeles1, González-Ramírez Luis A. 1, F. M. Baldock Peter2 , A. Briggs Richard2, A. Kolek Stefan2, D. Shaw Stewart Patrick2 , Gavira Jose A.1*
CONCLUSIONS
Protein Protein concentration and seed preparation
conditions
Micro-seeding conditions
Glucosa
Isomerase
20 mg/ml, Batch experiment using 100 mM
Hepes pH 7.0 and 0.9 M Ammonium sulfate.
30 mg/mL
10 mg/mL
10 mg/mL + seeds
10 mg/mL + dipped in seeds
Ribonuclease-A 240 mg/mL, Counterdiffusion technique,
precipitating solution C21**: 6 M Na-Formate.
240 mg/mL
50 mg/mL
50 mg/mL + seeds
50 mg/mL + dipped in seeds
Catalase 36 mg/mL, Vapor diffusion technique using the
crystallization mushroom. Precipitating solution
10% w/v PEG-4000.
40 mg/mL
7 mg/mL
7 mg/mL + seeds
7 mg/mL + dipped in seeds
Myoglobin 80 mg/mL, Counterdiffusion technique,
precipitating solution C1**: 30% PEG 4K, 0.2 M
Mg Chloride, 0.1 M Tris-HCl pH 8.50
80 mg/mL
5 mg/mL
5 mg/mL + sedds
5 mg/mL + dipped in seeds
α-Lactalbumin 72 mg/mL, Hanging drop technique.
Precipitating solution C23**: 0.82 M K-
Phosphate, 0.82 M Na-Phosphate, 0.1 M Na-
Hepes pH 7.50.
30 mg/mL
5 mg/mL
5 mg/mL + sedds
5 mg/mL + dipped in seeds
Thaumatin 30 mg/mL, D5 from Jena Bioscience Membrane
screen 3; 1.5 M Lithium Sulfate, 100 mM HEPES
Sodium Salt, pH 7.5
90 mg/mL
10 mg/mL
10 mg/mL + sedds
10 mg/mL + dipped in seeds
Xylanase 36 mg/mL, No.45 from Molecular Dimensions
Structure Screen1 ; 4.0M Sodium Formate
56 mg/mL
36 mg/mL
36 mg/mL + sedds
36 mg/mL + dipped in seeds
Trypsin 30 mg/mL, D6 from Jena Bioscience Membrane
screen 3; 2.0M Sodium Chloride, 100mM
Sodium Formate
90 mg/mL
30 mg/mL
30 mg/mL + sedds
30 mg/mL + dipped in seeds
Concanavalin 5 mg/mL, B8 from (Molecular Dimensions) JCSG
plus screen; 10% w/v PEG 8000, 100ml Tris-HCl,
pH 7.0, 200mM Magnesium Chloride
15 mg/mL
5 mg/mL
5 mg/mL + sedds
5 mg/mL + dipped in seeds
Figure 1. Protein crystals microphotographs. Capital letters show the seeds used,
small letter crystals after seeding. A) Glucosa Isomerase crystals obtained by batch 20
mg/mL and 100 mM Hepes pH 7.0 and 0.9 M Ammonium sulfate, a) Glucosa Isomerase
crystals after seeding 10 mg/mL condition C2** B) Catalase crystallized at 36 mg/mL by
vapor diffusion technique and 10% w/v PEG-4000, b) Catalase at 7 mg/mL condition
C8**, C) Myoglobin obtained at 20 mg/mL condition C1**; c) Myoglobin crystallized at 5
mg/mL condition C1**; D) Thaumatin 30 mg/mL and precipitated with D5 from Jena
Bioscience Membrane screen3; 1.5 M Lithium Sulfate, 100 mM HEPES Sodium Salt, pH
7.5; d) Thaumatin 30 mg/mL and obtained in condition C7**; E) α-Lactalbumin
crystallized at 72 mg/mL by Hanging drop technique and 0.82 M K- Phosphate, 0.82 M Na-
Phosphate, 0.1 M Na-Hepes pH 7.50.; e) α-Lactalbumin crystallized at 5 mg/mL. C13**;
F) Ribonuclease-A obtained at 240 mg/mL condition C21**; f) Ribonuclease-A
crystallized at 50 mg/mL condition C21**.
A a B b
C c D d
E e F f
**Counterdiffusion Screening Kit ® ref. GCB-CSK-24. See the last part appendix thecomposition of the 24 conditions.
TABLE 1. Crystallization conditions for initial protein micro-seeds preparation and the final condition to study micro-seeding.
1 2 3 4
0
1
2
3
4
5
6
7
8
Hits n
um
ber
Ribonuclease-A
240 mg/mL 50 mg/mL 50 mg/mL
+ seed
50 mg/mL + dipped
in seeds
1 2 3 4
0
1
2
3
4
5
6
7
8
Hits n
um
ber
Myoglobin
80 mg/mL 5 mg/mL 5 mg/mL
+ seed
5 mg/mL + dipped
in seeds
1 2 3 4
0
3
6
9
12
15
Hits n
um
ber
- Lactalbumin
30 mg/mL 5 mg/mL 5 mg/mL
+ seed
5 mg/mL + deeped
in seeds
1 2 3 4
0
2
4
6
8
10
12
14
Hits n
um
be
r
Tripsin
90 mg/mL 30 mg/mL 30 mg/mL
+ seeds
30 mg/mL + dipped
in seeds
1 2 3 4
0
5
10
15
20
25
Hits n
um
ber
Concanavalin
15 mg/mL 5 mg/mL 5 mg/mL
+ seeds
5 mg/mL + dipped
in seeds
1 2 3 4
0
2
4
6
8
10
12
14
10 mg/mL + dipped
in seeds
10 mg/mL
+ seeds
Hits n
um
be
r
Glucose Isomerase
30 mg/mL 10 mg/mL
1 2 3 4
0
2
4
6
8
10
12
14
10 mg/mL + dipped
in seeds
10 mg/mL
+ seeds
Hits n
um
ber
Thaumatin
90 mg/ml 10 mg/mL
1 2 3 4
0
2
4
6
8
10
12
14
16
18
20
Hits n
um
ber
Catalase
40 mg/mL 7 mg/mL 7 mg/mL
+ seed
7 mg/mL + dipped
in seeds
1 2 3 4
0
2
4
6
Hits n
um
ber
Xilanase
56 mg/mL 36 mg/mL 36 mg/mL
+ seeds
36 mg/mL + dipped
in seeds
TABLE 2. Number of hits in the protein crystallization study incapillaries by combination of counterdiffusion and micro-seedingtechniques using different model proteins.NOTE: The last capillary correspond to the filled with the proteins solution and after dipped in the Seed-Syrup Stock solution also named as “touch”.
For the first time, it is reported the crystallization of several model proteins using micro-seeding by the counterdiffusion technique. Overall, most of the protein studied (90%) increasethe number of hits in which the nucleation occurs even when the capillary only touches the“seeds syrup”.The most effective protocol was mixing the protein solution with the seed-syrup, which
produced an homogeneous distribution of seeds along the capillary.It is well known that the initial seed may influence definitively the quality of the final crystals.
However in this work the use of (in appearance) not so good quality crystals, such as the case ofspherulites of Myoglobine, was done on purpose resulting in the growth of faceted crystals.
Seeds from the initial crystalline-precipitate can be used to set-up a minimum counter-diffusion screening experiment, even at low protein concentration, to find new crystallizationconditions and to produce crystals of improved visual quality.
Distinct methods have being studied to improve quality andsize of protein crystals. Protein crystallization in capillaries bycounterdiffusion technique has been used routinely for crystaloptimization. On the other hand, seeding techniques has opena possibility to improve protein crystal quality and size bycarrying on new crystallization experiments on the metastableregion of the phase diagram where only growth can occur.Ideally, seeding should be implemented using batch technique
to avoid subsaturation conditions that could dissolve the seedor, high supersaturation conditions as can be obtained invapour diffusion technique. Counterdiffusion technique allowsthe introduction of seeds into a system where the masstransport is controlled by diffusion in which supersaturationincrease slowly enough to permit the growth of several seedsminimizing cross influence, mixing and conventions effects.In this work we present the proof of this concept producing
optimized crystals of several model proteins by the combinationof counterdiffusion and micro-seeding techniques.
• A. McPherson, in: Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press, New York, 1998 (Appendix section).
• Enrico A. Stura and Ian A. Wilson, (1990). Analytical and Production Seeding Techniques. METHODS: A Companion to Methods in Enzymology. Vol. 1, No.
1, August, pp. 38-49.
• J.M. García-Ruiz, (2003). Counterdiffusion Methods for Macromolecular Crystallization. Methods in Enzymology, Vol. 368 130-154.
• R.J. Read and J.L. Sussman (eds.): Evolving Methods for Macromolecular Crystallography, (Succeeding with seeding: some practical advice, Terese Berfors)
1–10 © 2007. Springer
• T. Bergfors, (2003). Seeds to crystals, Journal of Structural Biology 142, 66–76.
• E. Saridakis,K. Dierks, A. Moreno, M. W. M. Dieckmannd and N. E. Chayen. (2002). Separating nucleation and growth in protein crystallization using
dynamic light scattering. Acta Cryst. D58, 1597-1600.
ACKNOWLEDGEMENTS
E-mail: [email protected], [email protected], ** [email protected]
Seed-Syruppreparation
200 mL proteinsolution
+ Crystalline material
Crystals were crushed using a glass capillary
[protein] [protein] = CONTROL
1 2
Protein +
Seed-Syrup
Seed-SyrupStock
and capillary “touch”
3
4
Capillaries
1 2 3 4
Crystalline material
Spherules, amorphous
aggregates, needle or micro-crystals
aggregates.
Obtained by different techniques
(Bath, vapour diffusion,
counterdiffusion)
Counterdiffusion
Screening Kit ®
GCB-CSK-24 **.
One of the goals of this study was to increase the number of hits during the screening in capillaries by counterdiffusion using the minimum Kit (24 conditions) using micro-seeding technique. Our results show that both protocols, mixing the seed with the protein solution and touching the seed syrup, increase the number of hits when compare with the low concentration protein solution (90%). In three cases (33%) the number of hits using seeds was higher than those obtained with high protein concentration solution and in one case we got crystals only when seeds were included.
