Dear Gloria,
probably even when you make the protein synthetically there will be
proteases in your solution, because many "air allergens" are proteases
(The major HDM allergens (i.e., allergens recognized by the majority of
HDM allergic subjects) are present in high amounts in the fecal pellets
of these animals. The pellets become airborne particles that can easily
be inhaled, and contain bacterial components stimulating the innate
immune system [14
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535861/#B14-ijms-18-01368>].)
And even when you express without tag and purify from a biological
expression system without using a protease to cleave the affinity tag,
you will have proteases in your final protein solution. (Proteolytic
fragments of proteins have also been crystallized serendipitously. In
most cases, the purified protein or the crystallization solution had
been contaminated with trace amounts of protease and the proteolysis
occurred during crystallization. There are many well-characterized cases
of this occurring, for example, references7
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366506/#R7>and8
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366506/#R8>, and
doubtless there have been many more undocumented examples.)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366506/
So I think there is no simple answer to your problem. So I would go with
the quote"Dosis sola venenum facit" = "The Dose Makes the Poison"
Do people know which (kind of) proteases cleave receptors? So then
taking a very specific protease might be the best.
There is an autoproteolytic system out there which might be interesting
for you. The protein goes to inclusion bodies and after refolding the
fusion protein is cleaved off. As you know inclusion bodies have the big
advantage that the protein is already very pure before the "real"
purification is started.
"We describe a prokaryotic expression system using the autoproteolytic
function of N^pro from classical swine fever virus. Proteins or peptides
expressed as N^pro fusions are deposited as inclusion bodies. On/in
vitro/refolding by switching from chaotropic to kosmotropic conditions,
the fusion partner is released from the C-terminal end of the
autoprotease by self-cleavage, leaving the target protein with an
authentic N terminus. A tailor-made N^pro mutant called EDDIE, with
increased/in vitro/and decreased/in vivo/cleavage rates, has enabled us
to express proinsulin, domain-D of staphylococcal protein A, hepcidin,
interferon-α1, keratin-associated protein 10-4, green fluorescent
protein, inhibitorial peptide of senescence-evasion-factor, monocyte
chemoattractant protein-1 and toxic gyrase inhibitor, among others. This
N^pro expression system can be used as a generic tool for the high-level
production of recombinant toxic peptides and proteins (up to 12 g/l)
in/Escherichia coli/without the need for chemical or enzymatic removal
of the fusion tag."
It would be interesting for me to hear if somebody is using this system
regularly.
https://www.nature.com/articles/nmeth1116
https://www.ncbi.nlm.nih.gov/pubmed/18026112
https://www.ncbi.nlm.nih.gov/pubmed/23871287
If the protein is unstructured or refolds very easily adding harsh
reagent to kill the remaining proteases is probably the best what can be
done.
Br, Georg.
On 2018-09-20 22:17, Gloria Borgstahl wrote:
Hello, friends in crystallography,
A colleague just asked me this question. He is worried about trace
protease interfering with the receptors he is studying in cell-based
experiments using a 110 amino acid protein we made for him. He has
been unable to make the peptide synthetically. The company is having
trouble getting that to happen. Any ideas? Happy Thursday, G
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