Protein Expression, Purification, and Analysis

Question 1

Recombinant proteins

Answer 1

Made from recombinant DNA to bring together genetic material from multiple sources and create sequences that would not otherwise be found in the genome or in nature. They are commonly created to study the function of a particular protein. They are expressed in easy to culture organisms and purified so they can be analyzed

Question 2

How is recombinant DNA created?

Answer 2

Two pieces of DNA spliced together from different sources. Generally, a particular gene is spliced onto a plasmid. Restriction enzymes create “sticky ends” that allow for the insertion of the gene of interest. DNA ligase heals the breaks

Question 3

Expression/production systems (3)

Answer 3

1. E. coli- most common
2. Yeast
3. Mammalian cells

Question 4

E. coli recombinant production systems

Answer 4

Simple, convenient, rapid, and cheap. Proteins that require disulfide bonds may be an issue, but there are ways around this. The expression of some mammalian proteins can be challenging in a bacterial cell due to lack of appropriate machinery, including glycosylated proteins and tRNAs

Question 5

Yeast recombinant production systems

Answer 5

More challenging than E. coli, but better for eukaryotic proteins.

Question 5

Inducible expression

Answer 5

Turning on the transcription when you want it to turn on. This produces greater levels of recombinant target expression

Question 5

pET vector components (6)

Answer 5

Commercially available vectors. 1. Genes for resistance to the antibiotic kanamycin. The gene for kanamycin resistance is important because it’s necessary for selection of bacteria that harbor the plasmid. We want to select for bacteria with the plasmid because we want to express the recombinant gene on the plasmid.
2. Multiple cloning site, which has recognition sequences for multiple restriction enzymes. All of these restriction enzymes create single stranded overhangs- “sticky ends”. This is where the gene of interest is inserted
3. LacI- encodes the Lac repressor. It is the basis for how we turn transcription on/off
4. T7 promoter- needs the T7 RNA polymerase
5. Lac operator- the genetic switch the lac repressor binds to, regulates expression of the repressor
6. Histidine tag- usually located at the C terminus. Useful for affinity purification, histidine has an affinity for nickel

Question 6

BL21(DE3)

Answer 6

An E. coli expression strain that is deficient in Lon protease and OmpT protease. Don’t want a lot of proteolytic activity because we are trying to express a recombinant protein. Contains λDE3 lysogen, meaning that it carries the gene for T7 RNA Pol under control of lacUV5 promoter controlled by the lac repressor

Question 7

Isopropyl β-D-1-thiogalactopyranoside (IPTG)

Answer 7

A molecular mimic of allactose, which we add too induce expression of the recombinant protein. It will induce transcription of T7 RNA polymerase. We don’t use lactose because it could be metabolized by the bacterial cell

Question 8

Recombinant protein expression in E. coli - induction

Answer 8

When we don’t add IPTG, the lac repressor stays bound and we don’t get expression of the T7 RNA polymerase. When IPTG is added, it binds to the Lac repressor, causes it to release, and allows for expression of RNA polymerase. The pET28 vector, which contains the recombinant target gene, is also under the control of the Lac operon. If IPTG is not added, and RNA polymerase is not expressed, it will not bind to the promoter. At this point, the Lac repressor is still bound, providing an additional measure of repression. Adding IPTG causes the RNA polymerase bind to the T7 promoter, the lac repressor is no longer bound, and the recombinant gene can be produced.

Question 9

How is recombinant protein expression verified?

Answer 9

With western blot. If you don’t have a specific antibody for a target protein, you can also use an antibody with a histidine tag

Question 10

How are proteins gotten out of E. coli systems? (4)

Answer 10

1. Lysozyme- digests the peptidoglycan cell wall
2. Protease inhibitors- don’t want to lose the target protein
3. Nucleases- break up any nucleic acids
4. Cell disruption through sonication or detergents

Question 11

Differential centrifugation

Answer 11

We need to make sure that our recombinant protein is soluble- that is has the correct tertiary and quaternary structure. Differential centrifugation spins the solution at a high speed. Soluble proteins end up in the top fraction (supernatant) and insoluble proteins end up in the lower fraction. We can then test the supernatant for our protein, and if it is present, it is soluble

Question 12

What do we do if a recombinant protein is not soluble? (3)

Answer 12

1. Can adjust IPTG concentration- reduce IPTG to slow down expression
2. Can adjust growth temperature- cooling it down to slow down expression
3. Can adjust induction time

Question 13

When is a protein targeted to the E. coli periplasm?

Answer 13

If a protein requires disulfide bonds (like antibodies) or if it is a periplasmic protein. The E. coli periplasm is an oxidizing environment that promotes disulfide bond formation. It is more oxidizing than the cytoplasm. We can target proteins to the cytoplasm by adding a leader peptide

Question 14

Components of the Sec system (4)

Answer 14

1. Leader peptide- added to the recombinant protein
2. Chaperones & chaperone-like proteins
3. Membrane bound translocase
4. Cytoplasmic ATPase- not always necessary

Question 15

SecYEG complex

Answer 15

A spherical molecule forming a pore in the inner membrane. It is closed if no proteins are being transported through it. A seam opens to allow lateral diffusion of cleaved leader peptide into membrane

Question 16

Targeting recombinant proteins to E. coli periplasm (5 steps)

Answer 16

1. The leader peptide binds SecYEG, and the central pore opens
2. The leader peptide is cleaved by signal peptidase, and translocation is completed
3. The central pore closes
   4.SecYEG seam opens, and the cleaved signal sequence is transferred laterally into the hydrophobic interior of the membrane
4. The leader peptide is rapidly degraded

Question 17

Methods of protein translocation to the periplasm

Answer 17

1. Co-translational protein secretion
2. Post-translational protein secretion

Question 18

Co-translational protein secretion into the periplasm (6)

Answer 18

This process occurs at the same time that the ribosome is translating the protein.
1. The ribosome makes a protein with a signal peptide at the end terminus
2. Signal recognition particle (SRP) binds to the leader peptide. It also binds to the ribosome, between the large and the small subunit. This binding transiently stops translation
3. SRP binds to its receptor, in close proximity to SecYEG
4. Once they are in close proximity, the leader peptide takes over and directs the translating ribosome to SecYEG
5. The ribosome takes over to push the protein into the periplasm
6. Signal peptidase is an inner membrane enzyme cleaves the leader peptide

Question 19

How can we get proteins out of the periplasm?

Answer 19

Through periplasmic lysis

Question 20

Periplasmic lysis (periplasm prep)

Answer 20

The goal of this process is to disrupt the outer membrane and peptidoglycan, but leave the inner membrane and cytoplasm intact- we only want to release proteins from the periplasm, nothing should be released from the cytoplasm

Question 21

What is needed for periplasmic lysis? (3)

Answer 21

1. EDTA- metal chelator (magnesium)
2. Lysozyme- digests peptidoglycan cell wall
3. Sucrose- osmotically protect the inner membrane

Question 22

Post-translational protein secretion into the periplasm (2)

Answer 22

Occurs after the ribosome has made the protein
1. The SecA enzyme goes through repeated rounds of binding, hydrolysis, and release, undergoing conformational changes
2. The conformational changes push the protein across the SecYEG pore into the periplasm

Question 23

Which molecules help to conduct periplasmic lysis?

Answer 23

Magnesium is bound between LPS residues, creating a barrier in the outer membrane. Adding EDTA removes the magnesium, making the outer membrane more permeable. Lysozymes degrade the cell wall. Sucrose protects the inner membrane, preventing it from lysing and ensuring that only proteins will be leaving the cytoplasm

Question 24

Why must recombinant proteins be purified?

Answer 24

When proteins have been released from E. coli, the target recombinant protein is among the many proteins. Therefore, the recombinant protein must be isolated

Question 25

Column chromatography

Answer 25

Carries out protein purification using columns. What is in the column differs based on the type of purification we’re doing.

Question 26

Ni-Histidine affinity purification

Answer 26

Most recombinant proteins are made with a 6X His tag. His has a binding affinity for nickel, so a His-tagged protein will bind nickel in a column purification, but other proteins will not. In the end, you should just have your recombinant protein with the Histidine tag

Question 27

Nickel-Histidine interaction

Answer 27

Histidine has a side chain called an imidazole ring, and this side chain binds to nickel

Question 28

How is Ni-histidine affinity purification conducted?

Answer 28

We take the proteins that have been removed from E. coli and run them through a column coated in nickel. Other proteins flow through, but the recombinant protein has a histidine tag and will bind the nickel. However, some E. coli proteins may still have a high enough histidine content to bind to nickel, so we do several wash fractions using a low concentration of purified imidazole. The low concentration should out-compete the E. coli proteins but not the histidine tagged proteins. After this, we used a high concentration of imidazole for the elution step, removing isolated proteins from the column

Question 29

GST-glutathione affinity purification

Answer 29

Glutathione-S-transferase (GST) is added as a tag to a recombinant protein. This is a full protein (211 amino acids) as opposed to 6X His. GST rapidly folds into a stable and highly soluble protein upon translation, and often promotes greater expression & solubility of recombinant proteins. Binds glutathione (GSH), and you elute with reduced GSH

Question 30

Ion-exchange chromatography

Answer 30

A method of separating proteins based on their charge. This is useful for purifying native, endogenous proteins (they are not tagged with anything), and useful as an initial “clean-up” prior to affinity chromatography. The proteins are passed through either an anion exchange column or a cation exchange column

Question 31

Ion-exchange chromatography outcomes are determined by (3)

Answer 31

1. pH of buffer used
2. Salt concentration
3. pI of protein of interest

Question 32

Isoelectric point (pI)

Answer 32

The isoelectric point is the pH at which a protein does not have a charge. A pH above the isoelectric point will give the protein a negative charge, and a pH below the isoelectric point will give the protein a positive charge

Question 33

Ion-exchange chromatography columns (2)

Answer 33

1. Anion exchange columns- positively charged resin
2. Cation exchange columns

Question 34

Anion exchange chromatography

Answer 34

The column is lined with positive ions. The buffer pH is above the isoelectric point, giving the target protein a negative charge. As the proteins are passed through, they bind to the ions. For elution, we gradually decrease the pH of the buffer. We will eventually hit the PI of some of the other proteins, as well as the target protein, helping to elute the proteins

Question 35

Manipulating ionic strength- ion exchange chromatography

Answer 35

Another method of eluting proteins from the column. Proteins of different charges have different affinities (aka ionic strength). We can adjust ionic strength by changing the salt concentration, which can disrupt the binding interactions of the target protein and cause it to elute

Question 36

Cation exchange column

Answer 36

The column is lined with negative ions. If the buffer pH is below isoelectric point, the protein of interest will develop a positive charge and will bind to the matrix. We can elute with salt or by manipulating the pH until we get to the isoelectric point

Question 37

Gel filtration chromatography (size-exclusion)

Answer 37

This method of protein separation is based on the size and molecular weight of the protein. No tags are involved here. It is useful for purifying native proteins and as an initial “clean-up” prior to or following affinity chromatography

Question 38

Gel filtration chromatography outcomes are determined by

Answer 38

Mobility of the protein through the porous matrix

Question 39

How does Gel filtration (size-exclusion) chromatography occur?

Answer 39

A column is filled with porous beads. Larger proteins are not able to enter the pores of the beads, they travel around them and exit the column first. Smaller proteins are able to enter the pores at different rates based on their size, so they come out last