X-ray studies of recombinant anti-testosterone Fab fragments

The use of PEG 3350 in crystallization

Jarkko Valjakka (Corresponding Author), Ari Hemminki, Tuija Teerinen, Kristiina Takkinen, Juha Rouvinen

Research output: Contribution to journalArticleScientificpeer-review

12 Citations (Scopus)

Abstract

Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 Å. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.
Original languageEnglish
Pages (from-to)218 - 221
Number of pages4
JournalActa Crystallographica Section D: Biological Crystallography
Volume56
DOIs
Publication statusPublished - 2000
MoE publication typeA1 Journal article-refereed

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Immunoglobulin Fab Fragments
Crystallization
Testosterone
X-Rays
Protein Engineering
Osmolar Concentration
Glycerol
polyethylene glycol 3350
Buffers
Molecular Weight
Observation
Ligands
Antigens
Temperature
Proteins

Cite this

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title = "X-ray studies of recombinant anti-testosterone Fab fragments: The use of PEG 3350 in crystallization",
abstract = "Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 {\AA}. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.",
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year = "2000",
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language = "English",
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pages = "218 -- 221",
journal = "Acta Crystallographica Section D: Structural Biology",
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X-ray studies of recombinant anti-testosterone Fab fragments : The use of PEG 3350 in crystallization. / Valjakka, Jarkko (Corresponding Author); Hemminki, Ari; Teerinen, Tuija; Takkinen, Kristiina; Rouvinen, Juha.

In: Acta Crystallographica Section D: Biological Crystallography, Vol. 56, 2000, p. 218 - 221.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - X-ray studies of recombinant anti-testosterone Fab fragments

T2 - The use of PEG 3350 in crystallization

AU - Valjakka, Jarkko

AU - Hemminki, Ari

AU - Teerinen, Tuija

AU - Takkinen, Kristiina

AU - Rouvinen, Juha

PY - 2000

Y1 - 2000

N2 - Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 Å. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.

AB - Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 Å. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.

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SN - 2059-7983

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