Sciencemadness Discussion Board

extracting PDC protien

azo - 24-4-2008 at 02:56

I dont want to rehash old threads but i would like to understand more about alchohol dehydrogenase complex .
There is lots of information out there about different ways to extract pdc protien from c utilis which i have selected due to it being resistant to toxic substrates.
And has anyone had experience doing this that could put light on the best way to proceed foward.
!!! what in the hell is the differance between whole cell pdc and purified pdc.

any help would be very much appreciated as i no very little about protien chemistry

regards azo.:o

Sauron - 24-4-2008 at 07:27

Where's chemoleo? He's the resident biochemist.

You are talking about extracting an enzyme.

Sounds like a job for HPLC to me.

Preperative, nonmetallic HPLC at that like a Waters 650 Advanced Protein Purification System. PTFE pump heads so no metal ion contamination of the enzyme. The injectors are nonmetallic too.

BTW it's protein not protien.

Maya - 24-4-2008 at 14:29

ahem, if I may.

you need to completely homogenize , your liver sample I presume, with a blender or dounce homogenizer.
then, filter the stuff thru cheesecloth. centrifuge the supernatant at high speed and keep the supernatant discarding the pellet assuming your enzyme is in the cytoplasm. ppt with sat. ammonium sulfate. all at ice cold. then you need to do some
ion exchange chromatography on a big column. either anion exchange or cation exchange and elute your compound of interest ( I won't tell you which , anion or cation cause I ain't gonna look it up ). then check your fractions for which ones exhibit ADH activity by assay enzymatically ( a spectrophotometer is handy ). thats exactly how you do it . but I don't have the exact protocol. straight from 1st year biochem

forgot to add the centrifugation step, very important!

[Edited on 24-4-2008 by Maya]

roamingnome - 24-4-2008 at 16:16

Having just obtained an International 6000 RPM centrifuge i am again excited about purified PDC protein

Not sure if wheat germ PDC is the most hardy as compared to c utilis, but the ease at getting it seems unparalleled.

http://www.jbc.org/cgi/reprint/196/1/375.pdf

Improved purification of pyruvate decarboxylase from wheat germ...
http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1432-1033...

See you must lysis the yeast cells but with wheat germ you could have crude protein in a few hours.

azo - 24-4-2008 at 17:28

Thanks for the info maya i take it you mean ppt with ammonium sulfate untill the enzyme just starts to ppt and then centrifuge correct me if i am wrong. I suppose i need to no the net charge on the pdc enzyme whether it is anion or cation any help with this info would help a lot.

roamingnome like you said i dont no if pdc from wheatgerm is as hardy as c utilis but for what i am doing from all refs it seems that pdc from c utilis seems to be the best and they also state that whole cell pdc performs much better than purified pdc. Proberly due to the protection from toxic substrates from other cells which are not there on purified pdc correct me if i am wrong .
Thanks for the tip on wheatgerm i will do some research on it.
! And thanks for the replies



regards azo

chemoleo - 24-4-2008 at 18:15

Ok, first off HPLC is definitely not the method of choice, because HPLC works under conditions (i.e. acetonitrile,IPA or methanol with 0.1 % TFA) where proteins unfold, meaning that activity is lost due to loss of the native fold. Essentially you end up with a polypeptide of random structure rather than the defined original structure of the native folded enzyme.

So you have to use buffers - usually isotonic of some kind (50 mM K phosphate, 100 mM NaCl pH 7.5 springs to mind).
Most proteins are happy under these conditions.

Maya, the procedure using ammonium sulfate precipitation, is this out of a text book referring to ADH or PDC specifically or one of many purification protocols used for protein purifications in general? With ammonium sulfate, the target precipitates only at a certain AS concentration range, which is the basis for purification! So usually steps of 0-0.5, 0.5-1.0, 1.0-1.5 etc M (NH4)2SO4 (or finer, upon optimisation) are taken and the fraction that contains the protein (assayed by mass, activity, whatever) is then further purified....but this is old school biochem.

Whole cell vs purified - naturally the whole cell extract is better because it contains cofactors and other proteins that you may not have present in your purified extract.
Despite what biologists claim about knowing about a particular subject/reaction, much more is UNknown. A fact I found out the hard way. And there are many crap biologists/biochemists, and I freely admit they wouldn't survive in other sciences.

Anyway, I can check up ADH (alcohol dehydrogenase) and PDC (pyruvate decarboxylase) in i.e. Methods in enzymology, but in the meantime a patent search might useful.

If you give me the biochemical properties of ADH/PDC (MW, pI, solubility, etc), I can work out a purification scheme for you...

[Edited on 14-11-2009 by chemoleo]

Maya - 24-4-2008 at 23:49

<<<< Maya, the procedure using ammonium sulfate precipitation, is this out of a text book referring to ADH or PDC specifically or one of many purification protocols used for protein purifications in general? With ammonium sulfate, the target precipitates only at a certain AS concentration range, which is the basis for purification! So usually steps of 0-0.5, 0.5-1.0, 1.0-1.5 etc M (NH4)2SO4 (or finer, upon optimisation) are taken and the fraction that contains the protein (assayed by mass, activity, whatever) is then further purified....but this is old school biochem.>>>>>>>>>>


you know as well as I do that this is of course completely old school biochem. generalizing for all enzymes. No optimization given, as I said. find out empirically the correct AS conc. and cationic or anionic properties. I forgot to mention centrifuge before and after AS ppt.
What other way does a lay person have of success?

The way it is done nowadays usually destroys activity but is much simpler, just lyse the sample, run it on an sds gel, transfer the gel to a membrane and then western blot it. Or alternatively, after running it on a gel , then stain it, excise the band at the correct mol. wt. and run it on the LC/MS.
But thats 2nd year biochem and pretty much too far above the laypersons means

But then the guy said he wants the entire complex that it binds to it as well? So then the best way is to immunoprecipitate it with your antibody and
sephadex A/G or equivalent

azo - 26-4-2008 at 00:46

Well i have been called a lot of things before but never a lay person been layed a few times i hope this is what you ment .
Getting back to you chemoleo i will post the relevant info for you as soon as possible ! btw it don't seem that you are to optimistic about the said subject/reaction.

thank you very much for all the help.

MagicJigPipe - 8-5-2008 at 10:38

Definition of layman from Dictionary.com:

2. a person who is not a member of a given profession, as law or medicine.

As you can see, it's not an insult. It just basically means you do not have advanced knowledge in a certain subject.

zimaman - 7-4-2009 at 20:01

Quote: Originally posted by chemoleo  
Ok, first off HPLC is definitely not the method of choice, because HPLC works under conditions (i.e. acetonitrile, DCM or methanol with 0.1 % TFA) where proteins unfold, meaning that activity is lost due to loss of the native fold. Essentially you end up with a polypeptide of random structure rather than the defined original structure of the native folded enzyme.

So you have to use buffers - usually isotonic of some kind (50 mM K phosphate, 100 mM NaCl pH 7.5 springs to mind).
Most proteins are happy under these conditions.

Maya, the procedure using ammonium sulfate precipitation, is this out of a text book referring to ADH or PDC specifically or one of many purification protocols used for protein purifications in general? With ammonium sulfate, the target precipitates only at a certain AS concentration range, which is the basis for purification! So usually steps of 0-0.5, 0.5-1.0, 1.0-1.5 etc M (NH4)2SO4 (or finer, upon optimisation) are taken and the fraction that contains the protein (assayed by mass, activity, whatever) is then further purified....but this is old school biochem.

Whole cell vs purified - naturally the whole cell extract is better because it contains cofactors and other proteins that you may not have present in your purified extract.
Despite what biologists claim about knowing about a particular subject/reaction, much more is UNknown. A fact I found out the hard way. And there are many crap biologists/biochemists, and I freely admit they wouldn't survive in other sciences.

Anyway, I can check up ADH (alcohol dehydrogenase) and PDC (pyruvate decarboxylase) in i.e. Methods in enzymology, but in the meantime a patent search might useful.

If you give me the biochemical properties of ADH/PDC (MW, pI, solubility, etc), I can work out a purification scheme for you...


I am interested to know if you have made that procedure yet. If not, here are some properties of the proteins:

ADH:
M.W.: 38kD
pI: 6.79
charge at pH7: -1.4

PDC:
M.W.: 61kD
pI: 6.18
charge at pH 7: -6.2

I am interested in separating the 2 proteins in bulk and keeping the PDC containing portion. I wouldn't need to purify past that point. I just need to exclude ADH. The organism would be saccharomyces cerevisiae.

chemoleo - 8-4-2009 at 16:04


Re bulk separations from crude extracts, I'd first do ammonium sulfate precipitation (thereby getting rid of all the fats, sugars, glycans etc), and see if the two precipitate at different concentrations. They probably will. Then dialyse into a low salt buffer, pH 8.
Then, anion exchange (Q columns) at pH 8, see again if you can separate if it didn't separate during the previous step. Then, as a final step, you can do size exclusion to really polish the proteins...