Lesson 11
Expressing and Purifying Proteins
In the last lesson we learned how to obtain and/or design a DNA plasmid for our protein of interest and how to transform that DNA plasmid into E.coli cells. This week we will talk about how we express and purify the proteins encoded in that DNA. Proteins that we express in the lab are called recombinant proteins. Proteins can be expressed from yeast, human cells, or insect cells, but here we will focus on bacterial cells (E.coli) which are most commonly used for protein expression.
Expressing Recombinant Proteins
We left off at the last lesson (after performing a transformation) with an agar plate containing E.coli colonies which now contain plasmid with the DNA sequence for our protein of interest. Remember that this plate also contains the antibiotic that our plasmid has resistance too. This means that only the E.coli cells containing our plasmid will be able to grow on our plate.
In order to obtain enough protein to use for experiments we need to grow a lot of bacteria (especially if our protein of interest is a membrane protein because they have low expression levels and are difficult to purify). We will pick one colony from our plate (by scrapping it with a pipette tip) and add it to a small amount of rich media (5-100 mL). This media contains all of the necessary nutrients for the bacteria to grow. Again, it also contains the antibiotic our cells have resistance to so we know only cells containing our plasmid will grow. We will let the bacteria grow and divide in a warm shaking incubator (at 37°C) overnight. In the morning, we will see that the cells have grown to a high density (the liquid will be cloudy or opaque). We will add 5-20 mL of this overnight culture to a 1L flask (shown on the right) and allow that to incubate even longer (generally around 4 hours). Growing the overnight starter culture first gives the cells a "head start" before they are added to the larger flask.
Now that we have a lot of E.coli cells expressing the plasmid for our protein of interest, we will induce the cells, which tells the cells to start producing the protein encoded by the plasmid. The plasmids that we use contain a lac operon before the DNA of our protein. Normally, there is a lac represser bound to the lac operon that does not allow protein expression. We add IPTG (isopropyl β-d-1-thiogalactopyranoside, an analog of lactose that is unable to be broken down by the cells) which binds to the lac repressor and releases it from the lac operon allowing the transcription of our protein of interest. After adding IPTG we allow the cells to express our protein for 4 hours at 37°C, or overnight at 25°C.
After we allow the cells to produce our protein, we will centrifuge (or spin very quickly) the cell mixture. One model of a centrifuge is shown below.
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Centrifuging will pellet the cells at the bottom of the centrifuge tube and we can decant (or pour off) the liquid above the cell mass. We can then freeze the cells until we are ready to purify our protein.
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Purifiying Recombinant Proteins
The first step of purification is to lyse, or break open, the E. coli cells so that we can obtain the protein that we have engineered our cells to produce for us. First, we resuspend our cell pellet in a buffer solution. There are several ways to lyse including flash freezing, sonication (the application of high frequency sound energy through a probe), through the use of enzymes, or homogenization. Homogenization is what we typically use, and involves forcing the resuspended cell pellet through a very narrow space. The increase in pressure breaks open the cell membrane. The homogenization instrument is shown below.
After we have lysed our cells we will centrifuge our cell mixture to pellet the cell debris and unlysed cells. Our recombinant protein will remain in the supernatant (liquid). If we are purifying a membrane protein, we will need an extra step. For membrane proteins we will ultra-centrifuge (spin even faster) the supernatant to pellet the cell membrane. This membrane pellet can then be resuspended with buffer containing detergent.
Q1: Why do we add detergent to our buffer if we are purifying a membrane protein?
Now that we have our protein suspended in a buffer solution we need to purify it away from any other protein that may be in our sample. The purification process will depend on the specific characteristics of the protein we are purifying. Read section 4.1.3 of the Biochemistry textbook to learn about some of the common ways to purify proteins. Often, when we are designing the DNA for our protein we will add specific tags which can help us with the purification process. The most common tag is a his-tag which is a series of histidine residues (generally 6 making a hexahistidine tag) at the beginning or end of the protein sequence. Proteins with his-tags can be easily purified using ion metal affinity chromatography (IMAC). In this type of column, nickel or cobalt is chelated (bound) to a special metal chelating resin. When the protein containing the his-tag is passed over the column, the histidine residues will bind to the metal ion (which has a positive charge). Thus, our protein will be bound to the resin and all other proteins the cell had made will pass through the resin (called flow through). The column can then be washed with more buffer to ensure all other proteins have flowed through. An elution buffer is finally passed over the column containing imidazole which has a similar structure to histidine. The imidazole will compete with and replace the his-tag and the protein will be released from the column.
IMAC columns can be gravity flow columns, which means you pour the liquid on top of the column and it passes through the resin due to gravity (middle). The gravity flow column here contains cobalt resin (pink). Nickel resin is blue. We can also use instruments (right) which can be programmed to add the specific volume of buffers that we want and the column flow is due to a programed pressure.
SDS-PAGE Gel Analysis
During the purification process we need a way to analyze what proteins we have in our solution. We do this using gel electrophoresis (separation of macromolecules in an electric field). Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) is the most common gel system. SDS is a detergent that denatures (unfolds) the protein and coats the protein in a negative charge. The polyacrylamide gel has a characteristic pore size which allows smaller proteins to pass through the the gel faster than larger proteins. The result is the separation of the proteins in the sample by molecular weight. Generally a marker lane is also included which contains proteins of known molecular weights for comparison. There are several types of gel compositions and percent acrylamide which can be used to separate different size proteins more effectively.
Watch this video demonstrating all of the techniques we have learned in this lesson.
Q2: When growing and expressing a new recombinant protein it is necessary to optimize the conditions. Below is a gel of optimization for a 18 kDa protein. Which conditions are best for the expression of this protein?
Q3: Below is a purification gel for a 17 kDa protein. Is this protein pure in the elution lanes? Did all of the recombinant protein come off the column?
Q4: Below is a purification gel for a 16 kDa protein. Is this protein pure? Was this purification effective? What could be improved?
