Proteins are a bit like people. People have common
structural themes, but there are still differences. Luckily, proteins differ
between each other more than people. Purification requires picking out ONE out
of many.
Any protein based sample is a complex mixture, containing
both the things you want (your protein) and a bunch of things you don't
want to have. Before you start to plan the purification protocol it is always a
good idea to collect as much information as you can about your sample. Consider
characteristics such as
- Molecular weight
- Isoelectric point
- Solubility
- Stability
- Known Functions
Knowing these characteristics for your protein and
critical impurities (e.g. proteases, proteins or other biomolecules that may
bind to your protein of interest, isoforms etc.) will help you to plan an
efficient purification protocol, because you can use them for separating
proteins from each other..
Proteins differ in the number of charged groups on their
surface. They may have hydrophobic parts, affinity tag you have added or some
biospecificity for other molecules. They can also vary dramatically in size.
All of these properties and insights should be used in the design of the
purification protocol.
Using a combination of properties makes purification
efficient. This is a cornerstone of the CIPP purification strategy;
(CIPP means Capture, Intermediate Purification and Polishing). This is
always a good approach when you need to set up a purification protocol for your
protein since each chromatography technology has its own limitations
(we'll discuss in more detail in a coming post).
DHFR Example
In our DHFR project we hope
to show you the thinking behind our choices as well as sharing the
consequences.
So, before we start to plan the protocol for the
purification, we need to collect some information about DHFR.
So as suggested we started to plan the purification protocol by collecting some information
about DHFR.
The information we found
- it is a single chain enzyme involved in the process for synthesis of nucleic acids (DNA)
- it contains 186 amino acids and only one Cysteine, so there are no disulfide bridges in the structure.
- the polypeptide folding contains 8 beta sheets connected via 4 alpha helices
- the active site is situated in the N-terminal half of the sequence
- the molecular weight was 21.5 kDa and isoelectric point (pI) pI: 6.9
- it is a single chain enzyme involved in the process for synthesis of nucleic acids (DNA)
- it contains 186 amino acids and only one Cysteine, so there are no disulfide bridges in the structure.
- the polypeptide folding contains 8 beta sheets connected via 4 alpha helices
- the active site is situated in the N-terminal half of the sequence
- the molecular weight was 21.5 kDa and isoelectric point (pI) pI: 6.9
The pI is important for choosing which ion exchange chromatography
technique and which conditions that should be used in your protocol. The size of
the protein is important to know to choose a size exclusion chromatography
medium with a correct separation range.
To find the above information about the
structure of DHFR we use the data base UniProt/Swiss-Prot as a starting point (http://www.uniprot.org/) we
like this resource a lot because it is a hub for much of the information that
is known about most proteins.
Next the
interesting part of the analysis (and choosing which methods to use) which we'll discuss in an
upcoming post.
Remember the more you know the easier it is to
set up your purification protocol and by getting to know your protein and the
differences in its structure the better your purification. There are many databases that are excellent shortcuts to gaining valuable information on your protein and if you
have some recommendations of your own please let us know via the comments
section.
For more on how to simplify planning & execution of protein
purification download our free handbook
