Lab 2 - Recombinant Protein Expression in E. coli

Question 1

What are the tasks of this lab?

Answer 1

• Quantitation of log phase bacterial growth
• Protein expression induction
• Harvesting of bacterial cells by centrifugation
• Cellular lysis by sonication
• Bradford assay and preparation of samples for SDS-PAGE analysis

Question 2

What is the objective of this lab?

Answer 2

To induce the expression of a recombinant protein in E. coli

Question 3

What is important to understand prior to starting this lab?

Answer 3

How the expression of a cloned gene can produce a functional recombinant protein

Question 4

What is the first step to expressing a cloned gene to produce a functional recombinant protein?

Answer 4

Construct a functional gene expression vector

Question 5

What is the most popular cloning vector?

Answer 5

A plasmid

Question 6

What is a plasmid?

Answer 6

A plasmid is a circular DNA molecule which has the ability to replicate without the interaction of a host chromosome

Question 7

How is a genetically engineered plasmid introduced into a host system?

Answer 7

By a transformation process

Question 8

For this lab, what has already been constructed for you and how?

Answer 8

A recombinant expression vector by inserting the DNA sequence of the human phosphatase and tensin homolog (PTEN) into a pET vector

Question 9

What does the pET vector contain?

Answer 9

Both the transcriptional and translational elements necessary to regulate the gene expression of PTEN

Question 10

What does the pET-PTEN expression vector contain?

Answer 10

A well characterized promoter located upstream (3’) of the multiple cloning site

Question 11

What specific functional elements does the pET-PTEN expression vector contain?

Answer 11

• Ampicillin (Ap) resistance marker
• ColE1 origin of replication
• F1 origin of replication
• lacI gene
• T7 transcription promoter
• Lac operator up-stream of the promoter
• Multiple cloning site (where PTEN was inserted)

Question 12

In addition to the functional elements, what else does the pET-PTEN expression vector contain and why?

Answer 12

His6 tag located at the N-terminus of the PTEN sequence to allow for purification of the target protein utilizing affinity chromatography

Question 13

What are the transformed bacterial cells grown in?

Answer 13

Liquid media containing ampicillin

Question 14

What cells allow for ampicillin resistance and propagate?

Answer 14

Only cells containing the PTEN plasmid which expresses beta-lactamase

Question 15

What do the two origins of replication on the vector allow for?

Answer 15

Numerous copies of the vector to be produced

Question 16

What does the genetic engineering of the vector to contain a lac promoter (Plac) and associate lac operator region allow for?

Answer 16

The artificial induction of gene expression by the addition of a lactose analog, isopropylthio-beta-D-galactoside (IPTG) which binds to a transcriptional repressor (LacI) to allow the RNA polymerase access to the promoter

Question 17

What is the T7 transcriptional promoter designed to be specific for?

Answer 17

T7 RNA polymerase

Question 18

What is the polymerase derived from?

Answer 18

The host bacterium which contains the gene for T7 RNA polymerase integrated into its chromosome

Question 19

What can the lactose analogue IPTG induce?

Answer 19

The production of the target protein to 50% of cells total protein output within a few hours of induction by virtue of the Lac I allosteric mechanism

Question 20

What will we be exploiting in this lab?

Answer 20

A bacterial expression system in E. coli to produce large quantities of our target protein, PTEN

Question 21

What is the E. coli produced in milligram quantities of desired proteins used for?

Answer 21

• Enzyme assays
• Structure/function studies
• To produce antibodies

Question 22

What is the disadvantage of using a bacterial host system?

Answer 22

The lack of a mammalian post-translational modification system that may be imperative for proper folding and activity of mammalian proteins

Question 23

What bacterial cells line with be used in this laboratory?

Answer 23

BL21(DE3)-CodonPlus-RIL

Question 24

What are the BL21(DE3)-CodonPlus-RIL cells specifically engineered to contain?

Answer 24

Additional copies of rare tRNAs that recognize the Arginine codons, the Isoleucine codon, and the Leucine codon, enabling for improved heterologous protein production that may otherwise be restricted or limited in AT-rich genomes

Question 25

What happens when BL21(DE3)-CodonPlus-RIL lacks additional copies of rare tRNAs?

Answer 25

Can normally limit the translation of proteins in conventional E. coli strains such as the argU, ileY, and leuW tRNA genes

Question 26

What do the (DE3) cells offer?

Answer 26

An IPTG inducible system through the T7 lacUV5 promoter that is used in conjunction with T-7 promoter driven pET expression vector (pET-His6-PTEN)

Question 27

Why does the BL21(DE3)-CodonPlus-RIL cell line also contain a tRNA expression plasmid?

Answer 27

It is ColE1-compatible pACYC-based that houses an extra tRNA and copies and confers Chloramphenicol (Cam) resistance; which is required in the medium to maintain the pACYC plasmid

Question 28

Why does the BL21(DE3)-Codon-Plus-RIL cell line also have the enzyme endonuclease I (endA) inactivated?

Answer 28

To prevent the degradation of DNA in miniprep procedures

Question 29

Why does the BL21(DE3)-CodonPlus-RIL cell line also lack the long and ompT proteases?

Answer 29

Because they have been shown to degrade proteins during recombinant purification

Question 30

What do both lon and ompT proteases generated by E. coli degrade?

Answer 30

Abnormal and extracellular proteins to allow for better recombinant protein production and isolation

Question 31

Describe the procedure for the quantification of bacterial growth.

Answer 31

1) Acquire a 500 mL Erlenmeyer flask containing 200 mL of 2 x YT media that has been inoculated with BL21(DE3)-CodonPlus-RIL E. coli cells containing transformed His6-PTEN DNA
2) Transfer 1 mL from the His-PTEN bacterial culture and place into a labeled Eppendorf tube
3) From the aliquot of 2 x YT media, aspire 280 uL and place into well A1 of a 96-well plate
4) Repeat step 3 two times by adding 280 uL of media to wells A2 and A3
5) From the bacterial His6-PTEN culture you placed into the prelabelled Eppendorf tube, aspirate 280 uL of your His6-PTEN culture to well A4
6) Repeat step 5 two more times by adding 280 uL into wells A5 and A6
7) Load your 96-well plate to the Victor III plate reader
8) Ensure A600 value is approximately 0.6 and discard waste

Question 32

Describe the procedure for protein expression induction.

Answer 32

1) Aspirate and transfer 1 mL of uninduced 2 x YT His6-PTEN into a 1.5 mL microcentrifuge tube labeled “Total Cell Culture - Uninduced” and place on ice
2) Pipette 80 uL to 500 mL Erlenmeyer flask containing host cells (BL21) and transformed with His6-PTEN (also contains antibiotics and chloramphenicol)
3) Place the induced bacterial expression culture in a room temperature incubator (approx. 24 degrees C) equipped with a shaker platform with speed set at 175 rpm for induction to occur overnight (about 16 hrs.)

Question 33

Describe the procedure for protein isolation.

Answer 33

1) Acquire a new 500 mL Erlenmeyer flask of BL21 E. coli His6-PTEN liquid culture that has been previously induced with IPTG for 16 hours
2) Aspirate and transfer 1 mL of the induced 2 x YT His6-PTEN into a 1.5 mL microcentrifuge labelled “Total Cell Culture - Induced” and place on ice
3) Transfer the entire induced 2 x YT His6-PTEN culture into a sterile 250 mL centrifuge bottle
4) Use a balance to weigh and adjust the weight of two centrifuge bottles until their weight is identical
5) Have GA load two centrifuge bottles in a pre-chilled (4 degrees C) centrifuge and spin at 6,000 rpm for 15 minutes to pellet the cells
6) At the fume hood, carefully pour off the excess media
7) While centrifuge tube with pellet is on ice, resuspend the bacterial pellet in 1- mL of ice-cold cellular lysis buffer containing protease inhibitors
8) Transfer the bacterial cellular lysate into labelled 50 mL conical tube and keep on ice

Question 34

What is the procedure for cellular lysis by sonication?

Answer 34

1) Centrifuge “Total Cell Culture Uninduced” and “Total Cell Culture Induced” and centrifuge at max speed (15,000 rpm) for 2 mins at room temp
2) In fume hood, remove all supernatant and discard
3) Freeze the induced and uninduced pellets at 20 degrees C for lab 3
4) Keeping the 50 mL conical tube on ice, lyse the re-suspended bacterial pellet sample and insert the micro-tip of the Fisher Conicator Dismemberator into the tube to sonicate for 30 sec. and swirl the solution while on ice and let rest for 30 s
5) Repeat step 4 two more times
6) Transfer cample from conical tube into 50 mL Centrifugation Oakridge tube
7) Ensure tubes are of equal weight with those of another group and GA will spin them at 20,000 x g in a pre-chilled (4 degrees C) floor-mounted centrifuge for 15 mins
8) Transfer 200 uL to a 1.5 mL centrifuge tube labeled “Sonicate Lysate” and the remainder into a 15 mL centrifuge tube labeled “Sonicate Lysate”
9) Resuspend the pellet in the Oakridge centrifuge tube by vortexing 10 mL of 1 x PBS
10) Transfer 200 uL of resuspended pellet in 1.5 mL microcentrifuge tube labeled “Sonicate Pellet” and the remainder into a 15 mL centrifuge tube labeled “Sonicate Pellet”`

Question 35

What is the procedure for protein quantification by performing a Bradford Assay?

Answer 35

1) Pre-label 14 microcentrifuge tubes and add different volumes of Bradford Reagent and BSA protein standard (2 ug/uL), each with a final volume of 600 uL
2) Add different volumes of induced sonicate cell lysate and induced sonicate cell pellet into non-standard tubes, each with a final volume of 600 uL
3) Vortex samples and let stand for about 5 mins
4) Add 150 uL in triplicate of each into the designated wells of a 1/2 volume 96-well plate
5) Using the Victor III plate reader and WorkOut 2.5 software, measure the absorbance of your samples at 590 nm
6) Additional dilutions of samples may be required
7) Save samples at 20 degrees C for analysis in lab 3