20.109(S07):Bio-material engineering/Screening library

Introduction

Antibodies are amazing proteins, capable of recognizing almost any foreign substance we encounter. The overall structure of every antibody is identical. They are made from four polypeptides: two “heavy” (i.e. longer) chains and two “light” chains. The four chains assemble into the hallmark “Y” shape, with a disulfide bond holding each light chain to a heavy chain and another disulfide bond linking the two heavy chains. Yet each antibody has a unique “variable” region, at the tip of each Y, allowing for a specific interaction with a particular antigen. The variable region, called Fv for “variable fragment,” is defined by the sequences at the tip of the heavy chain and the tip of the light chain. You will be working with genetically engineered yeast that can display Fv regions on their surface, no small feat for a yeast since evolution has never required that yeast learn to make antibodies! To ease the task a little, the variable region of the light and heavy chains have been linked into a single polypeptide chain. Remarkably, a yeast cell can cram up to 100,000 copies of the single chain variable fragment (scFv) onto its surface.

To display the scFv protein on the yeast surface, a protein fusion was made between the scFv sequence and Aga2, a part of the a-agglutinin receptor that yeast normally express to help them mate. Two polypeptides, Aga1 and Aga2, are disulfide bonded to form the a-agglutinin receptor, with Aga1 embedded in the yeast cell wall. The Aga2:scFv fusion is expressed from a plasmid and the product is exported to the cell surface. It is anchored and displayed through disulfide links to the Aga1 protein.

Two additional sequences were engineered into the fusion. A short sequence called the HA-epitope was placed between the Aga2 and the scFv proteins, and another short sequence called the c-myc epitope was engineered at the C-terminal side of the scFv. These epitopes can be detected with commercially-available antibodies, and so they are useful for quantifying the amount of fusion protein at the cell surface.

The yeast express the Aga2:HA:scFv:c-myc fusion from a plasmid called pCT-CON. Other features of this plasmid include

• A yeast centromere
  • This allows the plasmid to replicate prior to each cell division
• The TRP1 gene
  • This allows yeast carrying the plasmid to be selected using –trp media
• The GAL1 promoter
  • This allows the display to be controlled by addition of galactose to the media
• A bacterial origin of replication and an ampicillin resistance gene
  • This allows the plasmid to be copied and genetically manipulated in bacterial cells

Yeast display plasmid

The yeast surface display system has been used to study protein:ligand interactions. Screens of antibody libraries have identified variants of pCT-CON that bind interesting ligands. Some of the novel pCT-CON variants that have been isolated bind other proteins (e.g. lysozyme, or the tumor antigen c-erb), others bind small molecules (e.g. fluorescein), and others even bind metals, as you will see today. Once a novel binder has been isolated, it is possible to mutate it further to identify new variants that bind with even greater affinity. This “affinity maturation” approach has been used to increase the affinity of one scFv:ligand interaction four orders of magnitude, changing the time required for the complex to dissociate from seconds to days! (Boder, Midelfort and Wittrup. PNAS 2000 97(20):10701-10705 [1]).

Today you will work with several yeast strains bearing either the original pCT-CON plasmid, that expresses an Aga2:scFv fusion that does not bind gold, or a variant called pAu1 whose AGA2:scFv product was selected based on its gold-binding ability. The yeast strain called “DY” (for Display Yeast) expresses the protein fusions when grown in galactose but not when grown in glucose. Strain “NDY” (for NonDisplay Yeast) are unable to present the fusion on the cell surface even in the presence of galactose since NDY do not carry the galactose-inducible form of the Aga1 protein to present the Aga2 fusions at the cell surface.

You should begin the gold panning protocol once you have counted or estimated the number of yeast colonies growing on the Petri dishes you prepared last time.

Protocol

Some of the work for this experiment has already been done for you. Two nights ago, the yeast strains needed for today were grown to saturation in glucose-containing media at 30°. A lack of tryptophan in that media insures that all the yeast carry the pCT-CON or pAu1 plasmids. Yesterday, an aliquot of the cells was moved to fresh media (“subcultured”). This time either glucose or galactose containing media was used. The cells on your bench have been grown at room temperature for at least 20 hours.

Panning for Gold

1. Prepare the gold slides for study by incubating them in Blocking/Binding Buffer. Wear gloves when you handle the slides and touch only the edges. Place a 6 mm x 10 mm slide into each well of a six well dish, shiny-side up. Add 2 ml Glu Blocking/Binding Buffer to the leftmost wells and 2ml Gal Blocking/Binding Buffer to the remaining four wells. Place the dish on the rocking shaker set at speed 7 and let it rock there for at least 10 minutes.
2. While the gold slides are blocking, move 5 ml of each strain to a 15 ml falcon tube. Be sure to swirl and fully resuspend the cultures before removing any cells.

  Tube     	Strain		Media
  1   		pCT-CON in DY	glucose
  2   		pAu1 in DY	glucose
  3   		pCT-CON in DY	galactose
  4		pAu1 in DY	galactose
  5		pCT-CON in NDY	galactose
  6		pAu1 in NDY	galactose

1. Harvest the cells by spinning the tubes in the centrifuge 2000 RPM, 5 minutes.
2. Aspirate the media from the cells. You do not have to remove every drop. In fact it’s better to leave a small amount of liquid on the cells rather than risk aspirating away the cells themselves.
3. Resuspend the cell pellet in 1 ml of the appropriate Blocking/Binding Buffer (Glu for the Glucose grown cells and Gal for the galactose grown cells).
4. Aspirate the Blocking/Binding Buffer from each of the gold slides and pipet 1 ml of fresh Blocking/Binding Buffer (again, Glu in the leftmost wells and Gal in the others).
5. Add the cells that you have resuspended into the appropriate well.

   

6. Place the dish on the rocking shaker set at speed 7 for 60 minutes. During this time you should read one of the available journal articles; before lab ends, you and your partner will present to the rest of the class the primary idea of the article and your thoughts on it. A two minute synopsis is all that is necessary, although more is fine too.
7. After panning for 60 minutes, move the gold slides to a new 6 well dish with 2 ml Glu or Gal Blocking/Binding Buffer. Use forceps to move the slides, touching only the edges, and wipe the forceps with ethanol between each sample.
8. Place the dish on the rocking shaker set at speed 7 for 30 minutes. During this time you will present your thoughts on the article you read and hear about the others.
9. Label six Petri dishes that have –trp media. They should be labeled with the same information that is on the six well dish as well as the date and your initials.
10. Use forceps to move the slides one last time into a new six well dish with 2 ml Glu or Gal Blocking/Binding Buffer.
11. Photograph the surface of the gold slides using the digital camera and the WILD® light microscope.
12. Move the gold slides to eppendorf tubes that have 500 ul of sterile water. Vortex each for 30 seconds exactly. Plate 100 ul on –trp Petri dishes. Once all your plates have dried, wrap them with your colored tape and place them in the 30° incubator, media side up.

DONE!

For next time

1. Create a table like the one below and make the required predictions (Y for yes, N for no, or NA if the experiment wasn’t done)

	Surface display of scFv fusion?		Binding to gold?
	Glucose	Galactose	Glucose	Galactose
pCT-CON in DY
pAu1 in DY
pCT-CON in NDY
pAu1 in NDY

1. Make a figure of your digital photographs, organizing them in the same order as your six well dish was organized. Be sure to include labels and a short description of the experiment. Include enough information so a person who didn’t do the experiment would understand the figure, but not a step-by-step protocol.
2. If you don’t know already, find out why BSA and Tween were included in the Blocking/Binding Buffer.
3. In preparation for the library screen you will perform next time, please review the chemistry involved in joining nucleotides during DNA replication. Draw the addition of a guanine nucleotide to a thymidine dimer, clearly indicating the 5’ and 3’ end.

Reagents list

Glu Blocking/Binding Buffer

• CAA(Glu)-see reagents list from last time
• 5 mg/ml BSA
• 0.1% Tween-20

Gal Blocking/Binding Buffer

• CAA(Gal)
• 5 mg/ml BSA
• 0.1% Tween-20