Nano seeding tools to generate nanometer size crystallization seeds

Patent metadata

Field	Value
Publication number	US-9901844-B2
Application number	US-201514640805-A
Country	US
Kind code	B2
Filing date	Mar 6, 2015
Priority date	Mar 7, 2014
Publication date	Feb 27, 2018
Grant date	Feb 27, 2018

Abstract

A kit and a method for using the kit to generate nanoseeds from protein nanocrystals and aggregates is disclosed. The method comprises mixing a plurality of beads with a protein nanocrystal or aggregate, and agitating the mixture to generate the nanoseeds. Nanoseeds made by disclosed embodiments may be of a high quality, as evaluated by TEM, and can be used to produce high quality protein crystals. Additionally, spectroscopic techniques, such as UV fluorescence and/or brightfield microscopy can be used to identify aggregates suitable to produce nanoseeds.

First claim

I claim:

1. A method, comprising:
contacting a plurality of beads with an aggregate comprising at least one first protein nanocrystal to form a mixture; and
agitating the mixture to produce a nanoseed.

2. The method of claim 1 , wherein the first protein nanocrystal has a diameter of less than 5 μm.

3. The method of claim 1 , wherein each bead independently has a bead diameter of less than 5 mm.

4. The method of claim 3 , wherein the bead diameter is selected from 0.1 mm, 0.5 mm, 1 mm or a combination thereof.

5. The method of claim 1 , wherein the plurality of beads is from 2 to 1000 beads.

6. The method of claim 5 , wherein the plurality of beads is from 2 to 100 beads.

7. The method of claim 6 , wherein the plurality of beads is from 5 to 30 beads.

8. The method of claim 1 , wherein the beads are glass beads, stainless steel beads or combinations thereof.

9. The method of claim 1 , wherein the aggregate is contained within a crystallization drop.

10. The method of claim 1 , wherein contacting a plurality of beads with an aggregate comprises contacting the plurality of beads with the aggregate in a microcentrifuge tube.

11. The method according to claim 1 , wherein agitating the mixture comprises vortexing the mixture, shaking the mixture, stirring the mixture or any combination thereof.

12. The method of claim 11 , wherein agitating the mixture comprises agitating the mixture for a time of from greater than 0 seconds to 30 seconds.

13. The method of claim 12 , wherein the time is from 5 seconds to 15 seconds.

14. The method of claim 1 , wherein the aggregate is a granular aggregate.

15. The method of claim 1 , further comprising selecting the aggregate by brightfield microscopy, transmission electron microscopy, or a combination thereof.

16. The method of claim 15 , further comprising screening the aggregate by ultraviolet tryptophan fluorescence.

17. The method of claim 1 , further comprising generating a second protein nanocrystal from the nanoseed.

18. A method, comprising:
selecting a plurality of aggregates by brightfield microscopy, each aggregate comprising at least one protein nanocrystal having a diameter of less than 5 μm;
contacting the plurality of aggregates with a plurality of beads having a diameter of from 0.1 mm to 1 mm, to form a mixture; and
agitating the mixture for from 5 second to 15 seconds to form a plurality of substantially homogeneous nanoseeds.

Assignees

• Univ Of Pittsburgh—Of The Commonwealth System Of Higher Education

Inventors

• Calero Guillermo A

Classifications

• B01D9/0077 Primary

  Screening for crystallisation conditions or for crystal forms · CPC title

• B01D2009/0086

  Processes or apparatus therefor · CPC title

• B01D9/0036

  Crystallisation on to a bed of product crystals; Seeding · CPC title

• B01F33/25

  Mixers with loose mixing elements, e.g. loose balls in a receptacle · CPC title

• B01F33/251

  using balls as loose mixing element · CPC title