Traditional Protein Purification vs Our System: Ideal Protein Purification

The ability to quickly and cheaply generate purified proteins is an absolute necessity for aspiring synthetic biologists and iGEM participants alike, since purified proteins are used in many key laboratory processes. Though some commonly used proteins (like EcoRI, XbaI, SpeI, and PstI) can be bought, many applications require synthetic biologists to purify their own target proteins either to study the activity of that protein or to use it in some other biological application. Many of us have personal experience purifying protein and have found it to be a slow and tedious process. For these reasons, the UW 2009 iGEM team has designed an Idealized Protein Purification (IPP) scheme that will speed up protein purification, and has the potential to make previously difficult-to-purify proteins easier to generate, in the hopes of making protein purification faster, and more accessible, to synthetic biologists.

Traditional Protein Production and Purification

Recombinant protein purification is a method whose basic steps have not changed significantly in at least the last 20 years - thus we believe this process is due for a rehaul. Traditional protein purification steps require expensive and specialized equipment (such as an ultracentrifuge and columns that bind to His tags) that may not be accessible to the average aspiring synthetic biologist. Traditional protein production and purification typically consists of the following three protein production steps and six purification steps:

• TRADITONAL PROTEIN PRODUCTION

1. The first step of traditional protein purification schemes is to clone in a favorite protein (referenced here as afp) into an expression plasmid designed to generate lots of protein.
2. Transform this plasmid into an expression strain of bacteria specialized in protein production.
3. Culture cells and induce protein expression allowing cells to grow to high density.

• TRADITIONAL PROTEIN PURIFICATION

1. Pellet Cells and re-suspend in a lysis buffer and incubate to ensure full cell lysis, releasing proteins trapped within the cell into the media (or break cells in a French Press).
2. Low-speed spin to get rid of cellular debris (membranes and such).
3. Long, high-speed spin media down in an ultra-centrifuge to remove remaining insoluble matter (membrane, insoluble proteins, etc) and leave soluble proteins in the supernatant.
4. Filter the supernatant to remove any lingering debris before being added to an affinity column. This ensures that the columns do not get clogged.
5. Run the filtered supernatant over an affinity column several times to ensure thorough binding of afp to affinity column.
6. Flow wash buffer over column to eliminate proteins that non-specifically bind to the column.
7. Collect afp by running an elution buffer over the column to release afp.

Though not necessarily hard, the purification process is tedious and time consuming, taking us about three and a half hours to complete on purification day. With this in mind, we created an idealized protein purification scheme that makes protein purification facile and cheaper, and takes a fraction of the time older methods require.

Idealized Protein Production and Purification

To avoid all the tedious purification steps one runs into during traditional protein purification we designed a system (the Ideal Protein Purification) in which the cell, not the researcher, performs the purification steps to make recombinant protein product. Ideal Protein Purification reduces purification to two easy steps (cloning remains the same):

• IDEALIZED PROTEIN PRODUCTION

1. Clone afp into a "target vector". This creates a fusion protein in which a "display tag" (called the NanoTag) is fused to the to the N-terminus of afp, and a "secretion tag" is fused to the C-terminus of afp.
2. Transform the target vector with the afp insert into cells already containing two additional plasmids. The first is a "Secretion plasmid", which contains all the parts of a type 1 secretion system from Erwinia chrysanthemi that specifically recognizes proteins that harbor the secretion tag and exports them outside of the cell. The second is a "Display plasmid" which expresses a protein that is directed to the cell surface and can bind to the "display tag", and then subsequently release the display tag when a specific small molecule is introduced to the system.
3. Grow up cells in our specialized cell line that contains both Secretion and Display plasmids and induce the expression of afp, just as in the traditional protein purification method. As the protein is produced it gets secreted to the media via the secretion system, and then binds to the outside of the cells via the display system. Now your protein of interest is non-covalently attached to the outside of the cell.

• IDEALIZED PROTEIN PURIFICATION

1. Spin down the cells and re-suspend in an elution buffer containing a small molecule which disrupts the interaction between the display tag and the protein it was binding to on the surface of the cells. This causes afp to be released from the surface of the cell.
2. Collect afp by pelleting the cells again and keeping the supernatant, which contains your purified protein.

This process should take about 10 minutes, quick enough to do between classes! In addition to saving time, our idealized protein purification method has other potential advantages over older methods. By keeping the cells intact, contaminants are not released into the media, which should result in purer protein product. A host of proteins normally toxic to . coli in the traditional protein purification system could be grown in the Ideal Protein Purification system since they would be secreted before reaching high intra-cellular concentrations. Alternately, some proteins cannot properly fold in E. coli, but by secreting these proteins into the appropriate media, they might fold correctly. Finally, there are some environmental applications, such as detoxification, where it is desirable to have a bacterium capable of secreting protein into the environment, which our system could be easily modified to do.