Confused about the different principles to freeze & thaw cells and purified protein

[Xuan Yang]

Dear Matt,

Thanks for the great & indepth explanation! You must be very good at
physics.

Interestingly, it was obvious that some proteins were born tougher than
others and could tolerate freezing and thawing process much better. Since
the freezing process seemed like a process of supersaturation, I was
wondering whether a better tolerance would indicate a better chance to form
crystals.

Moreover, Prof. Terese M. Bergfors mentioned in the book "Protein
Crystallization" that "Cells or bacteria tolerate freezing (-70ºC) better
than many purified proteins", as a result, it would better to store
expression hosts rather than purified proteins. And since cryopreservation
was cell/tissue type dependent, I was wondering whether anyone would like to
share some nice strategies to store expression hosts, especially E.coli.

Thanks in advance!

Sincerely,

Xuan Yang

2009/7/24 Matthew Connelly <MConnelly from lab901.com>

> When you freeze aqueous solutions slowly you form larger ice crystals. This
> would segregate water away from proteins and into the crystals of ice. As
> the amount of unfrozen water drops the protein becomes more and more
> concentrated and loses the water shell required to maintain its structure
> (remember the primary force for protein structure is the organisation of
> hydrophobic and hydrophilic residues). This quickly leads to protein
> denaturation and aggregation, and is one of the reasons why freeze thaw
> cycles are so damaging to proteins.
>
> Proteins are frozen quickly to produce the smallest ice crystals possible
> as quickly as possible, which minimises the disruption described above.
> Formulation of the protein with excipients such as polyols and carbohydrates
> can also help dramatically improve stability during freeze thaw by helping
> to lower the total amount of water bound up in ice crystals, and to take the
> place of water at the proteins surface. This can allow you to freeze
> proteins more slowly without causing damage.
>
> Not sure about cells, but I think that the freezing rates necessary for
> cryopreservation are actually dependent on the cell/tissue type...
>
> Matt
>
>
> -----Original Message-----
> From: methods-bounces from oat.bio.indiana.edu [mailto:
> methods-bounces from oat.bio.indiana.edu] On Behalf Of Xuan Yang
>  Sent: 24 July 2009 03:25
> To: methods from magpie.bio.indiana.edu
> Subject: Confused about the different principles to freeze & thaw cells and
> purified protein
>
>  Dear Sir or Madam,
>
> When we store cells (especially eukaryotic cells), storage box filled with
> isopropyl alcohol was used to prevent the temperature from dropping too
> fast. While thawing the cells from liquid nitrogen, we put the tube
> immediately into 37ºC water bath. Freezing slowly, thawing fast. This
> strategy was totally contrary to the way we deal with purified proteins,
> namely freezing rapidly (favorablely in liquid nitrogen), but thawing
> slowly
> (favorablely on ice). It was quite confusing and I was wondering whether
> anyone would be so kind to offer me some explanations.
>
> Sincerely,
>
> Xuan Yang
>
> National Laboratory of Biomacromolecules and
> Center for Infection and Immunity,
> Institute of Biophysics,
> Chinese Academy of Sciences,
> Room 1617, 15 DaTun Road,Chaoyang District,
> Beijing, China, 100101
> Tel: 86-10-64884329
> We will either find a way or make one.
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