Lab 5: Affinity Protein Purification of His-tagged Recombinant Proteins

Lab objectives

• Understand the basics of expression vectors and why we might want to purify a protein

• Understand how proteins are tagged

• Understand how a protein tag can be used for purification

• Understand the pET vector expression system

• Describe differences in bacterial strains that affect how we use them

What are we doing in this lab?

We’re purifying our expressed spider silk proteins out of a “lysate” that contains a mixture of all the other expressed bacterial proteins

Expression vector

A plasmid designed not only to carry and copy DNA, but to make the cell produce the protein encoded by that DNA

• Is able to replicate within bacterial cells, but also has unique features that make it specifically useful for actually expressing different proteins in bacteria

• Contains an inducible bacterial promoter

Inducible bacterial promoter

A DNA sequence that acts like a controllable on/off switch for a gene in bacteria

• Promoter is recognized by the bacterial transcription machinery

What does it mean for the promoter to be inducible?

We want to be able to control when the promoter is active (and the gene is being transcribed)

Why do we want the promoter to be inducible?

When bacteria express a gene from another organism, the bacteria can get sick and start to die

• If we control when gene expression happens, we can transform bacteria with our clone, keep the clone from being expressed, and know that we will not harm the bacteria

• We can grow large quantities of transformed bacteria, then induce expression of our gene of interest

• Even if protein produced by gene makes bacteria sick, we will have them make enough protein for us to collect before they die

What type of promoter does the pETBlue-1 vector contain?

A T7 promoter that controls the expression of our gene of interest

T7 promoter

• Originally identified from a bacteriophage

• Recognized by the RNA polymerase called T7 RNA polymerase

T7 RNA polymerase

A very simple RNA polymerase, containing only one subunit

• Gene that codes for T7 RNA polymerase has been inserted in the host E. coli chromosome

• Under the control of the lactose promoter and operon

What is the region that the T7 RNA polymerase-encoding gene has been inserted into?

DE3 region

What controls the expression of the T7 RNA polymerase gene?

Lac repressor protein

Lac repressor protein

• Binds to a regulatory sequence (“LacO”) on the host chromosome

• Blocks transcription of the T7 RNA polymerase gene, preventing its expression until lactose is present

How can we induce expression of our gene of interest?

By growing our transformed bacteria in media that contains an analog of the sugar lactose (IPTG)

How does lactose (IPTG) play a role in inducing expression of our gene of interest?

• Lactose binds to the lac repressor protein

• Causes the protein to change shape and fall off the DNA

• Allows initiation of transcription of the T7 RNA polymerase gene and subsequent expression

What happens after T7 RNA polymerase is made?

It diffuses within the cytoplasm of the bacterial cell and binds to the T7 promoter on your plasmid

• Transcribes spider silk gene

• Translated into the recombinant protein

Why do we want to purify the expressed spider silk proteins?

• We want to study the biochemical properties of a protein

  • Might want to see if a protein can bind to a specific sequence of DNA, so we could purify it to test for DNA binding

  • Might want to see if our protein has a specific enzymatic activity, ex. can it act as an ATPase, kinase, or protease?

  • Might want to make an antibody that recognizes it if we want to visualize the protein inside a cell

Can we purify proteins directly from the cells/tissue/organisms that produce them?

Yes, but it takes a lot of time and effort to figure out the unique properties of a protein that will allow you to purify it away from all the other proteins made in the cell or organism

• This type of purification can take a lot of time to figure out and many steps

If proteins are expressed at very low levels, it can be challenging to get enough of it to work with. How does our protocol work around that?

By expressing our spider silk proteins in bacteria, which are easy to culture, we can grow up significant amounts of the protein in our labs

How are we going to purify our specific protein of interest away from all the bacterial proteins?

We will use the 6xHis tag attached to the end of the spider silk protein

Protein tags

Tags are made from DNA sequences encoding various polypeptides that will be transcribed along with our gene and added to either the 5’ or 3’ ends of the transcript, or both

• Tags either the N- or C-terminal ends of our protein

• Many expression vectors encode the information needed to add protein tags to our protein of interest

6xHis tag

Tag is added by a reverse primer during PCR, using a similar approach to adding restriction sites to the 5’ termini of the amplified product

• PCR primers used to amplify the spider silk gene were designed so it was amplified to incorporate a 6xHis tag into the 3’ end of the gene in-frame with the spider silk gene codons (corresponds to the C-terminus of the protein)

  • Reads as spider-silk codons | His codon | His codon | His codon …

• Researchers built the His tag into the PCR product itself rather than adding it later

What happens after the spider silk cDNA was inserted into the pET24a cloning vector?

Placed adjacent to a Shine-Dalgarno sequence (bacterial ribosomal-binding site)

• Shine-Dalgarno sequence is short sequence on bacterial mRNA that tells the ribosome, “bind here, then start translating at the nearby start codon”

• Next to the ribosome binding site (RBS) will be your start codon (ATG) that will be used to begin translation

What happens when the spider cDNA gets expressed?

Translation of the mRNA will start at the AUG and read into the spider silk codons and finally into the 6 histidines in the C-terminus

• Your protein has been “tagged” with 6xHis tag

• Tag does not interfere with the structure or function of the purified protein

What are some examples of protein tags?

• 6xHis

• myc

• HA

• FLAG

All of these are small sequences of amino acids (some taken from sequences of other proteins, like HA-tag which contains part of the hemagglutinin protein)

Some vectors allow you to attach small functional proteins to your polypeptide—what does that do?

Can help increase protein stability in case your protein proves to be very unstable

• Ex. thioredoxin

What is the main reason why we want to tag a protein?

To help us visualize our protein in order to confirm that we are actually expressing it because we can purchase reagents that will allow us to visualize if our protein is actually being transcribed and translated

• Can also use tags to help us purify the protein for later experiments

What is the significance of the 6xHis tag we added to our spider silk proteins?

Helps us quickly purify large amounts of spider silk proteins from the bacterial cells without having to discover the unique properties of each protein

• Without a tag, your spider silk protein would look like just one protein among thousands of bacterial proteins

• You intentionally give your spider silk protein a feature that is easy to recognize, rather than finding a natural feature that makes it different from all the others to isolate it

Affinity chromatography

A purification technique that separates a target protein because it has a specific binding interaction with something attached to a solid support

• 6xHis tag has affinity for certain metal ions (ex. nickel and cobalt) and will bind to the metal ion (causing entire protein to stick)

• Most other proteins are not attracted to these metals, so they will not stick (and it’s really rare to naturally find 6 histidines in a row in a polypeptide)

What’s the significance with affinity chromatography and 6xHis tag?

The 6 histidines can bind to nickel ions, and the nickel ions can be attached to a number of different kinds of substances that help us capture our protein of interest

• We will be using nickel ions attached to magnetic beads

Explain the idea behind the mechanism of our protein purification

• Our protein of interest binds to the nickel ions

• The nickel ions are attached to magnetic beads

• We can use the magnet to grab the beads and pull our protein away from all the bacterial proteins present in the bacterial cell extract

Essentially: Bind target protein to beads —> magnet keeps target protein while you remove junk —> wash —> release target protein into a clean tube

What do we first do in purification? What comes after?

Lyse our bacteria to release their proteins (the “lysate”) and then add our bacterial lysate to the nicke beads

• 6xHis-protein binds to the nickel, and then we wash the beads to remove unbound bacterial proteins

• We need to elute our protein from the beads

How do we elute our protein from the beads?

Using the compound imidazole, which competes with the 6xHis tag for binding to the nickel

• Excess imidazole binds to the nickel and displaces the tagged protein

• We can now collect the purified protein

What do you do when your protein unfolds in the bacteria and clumps together, or is made at such high levels that it clumps together?

You lyse the bacteria in a buffer that is very strong and denatures your protein along with everything else in the bacterial cell

• You can still purify it, but it is probably not going to be functional

• That’s why we would add a thioredoxin tag to prevent this

We want to study the function of our protein, so what should we do?

Lyse the bacteria very gently and collect your protein in a buffer that allows you to keep the protein in its native (non-denatured) form

What is the strain of E. coli that we used in Lab 4?

JM109 cells

• Good for replicating plasmid DNA

What is the strain of E. coli that we’re using for Lab 5?

BL21 DE3 pLysS

• Good for purifying our protein

• Cells don’t contain mutations in the endA or recA genes, but instead have a mutated, inactive version of the RNase E enzyme, which protects the mRNA encoding your gene from being degraded

• Contains mutant, inactive forms of cellular proteases (ex. lon and OmpT proteases) which helps to limit the amount of degradation of our own protein of interest when we induce its expression

How were the E. coli strains transformed from one to another?

• Took some of the DNA from a positive clone identified via restriction digestion (last lab) and transformed DNA back into BL21 DE3 pLysS bacterial cells

• Next day, transformed cells were added to liquid LB broth + ampicillin

• Allowed to grow for 6-8 hours

• After, cells were treated with fake lactose (IPTG) to induce expression from promoter