MICR5831 L9: Protein Expression, Purification & Analysis 8/7/25 NEEDS SAQ

Describe two strategies for purifying proteins from cloned genes (slide 4)

SAQ

What are the characteristics of a fusion vector? (slide 5)

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Describe the lactose metabolism operon (slide 7)

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What happens to the expression of the lacZ operon in the presence of low levels of lactose? (slide 8)

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What happens to the expression of the lacZ operon in the presence of high levels of lactose? (slide 9)

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How does IPTG work to change the expression of a lacZ promoter? (slide 9)

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What are the important genetic features of pMALC2? (slide 11)

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Explain the blue white screening mechanism in the pMalC2 vector. (slide 12)

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What is the process for purifying MBP-fusion proteins? (slide 13)

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How does the polymerisation of acrylamide occur? (slide 16)

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Using a diagram, explain the organisation of a SDS-PAGE electrophoresis gel and apparatus. (slide 17)

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What is the purpose of SDS in SDS-PAGE? (slide 18)

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What is the purpose of mercaptoethanol in SDS-PAGE? (slide 18)

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Explain native and SDS PAGE and their relative advantages/disadvantages (slide 19 and 20)

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Using a diagram to illustrate, describe an outline for how Western blotting is performed. (slide 21 and 22)

SAQ

Why would purifying recombinant proteins be useful?

1) Rational design of drugs

2) Vaccine development

3) Study biological activity/function

4) Study protein structure

How can you use recombinant protein purification to rationally design drugs?

-Inhibit protein function/activity → kill microbes or inhibit mechanisms of pathogenesis

-EX: Neuraminidase inhibitors such as Relenza® (influenza)

Name some recombinant vaccines that require protein purification to work

-Hepatitis B vaccine

-Pertussis toxoid vaccine

What are two strategies for expression and purification of recombinant proteins?

1) Expression Vectors

2) Expression Vectors + Purification Vectors (Fusion)

What is this strategy for recombinant purification?

1) Expression Vectors

-Over-expression of proteins

-Chromatographic purification of proteins

-Requires specialist expertise, laborious

-Protein will have its original form/function

What is this strategy for recombinant purification?

2) Expression + Purification Vectors (Fusion Vectors)

-Overexpression and affinity chromatography

-Recombinant protein fused to a protein "tag"(fusion partner) with affinity to a specific substrate

-Relatively simple, no expertise required

-Fusion partner should be removed for protein to regain normal function

What are some general characteristics of Expression Vectors?

-Medium to high plasmid copy number (>20 copies/cell)

-Multiple cloning site (mcs)

-Strong regulated promoters

Why is it advantageous for Expression Vectors to have medium-high plasmid copy numbers?

-Easy to extract from host & manipulate

-Multiple copies → higher protein expression

Why is it advantageous for Expression Vectors to have an Multiple Cloning Site (MCS)?

-Flexibility in choice of restriction sites

-Allows directional cloning

Why is it advantageous for Expression Vectors to have strong regulated promoters?

Ptre or Ptac→ high level transcription → high level expression of protein

What are some other strongly regulated promoters besides Ptrc and Ptac with high levels of protein expression?

T7 RNA polymerase/promoter

AraC/Para

What is this?

-2 hexameric sequences at - 35 and -10 positions relative to transcription start point

-Separated by 15-20 bp

-Bind RNA polymerase → initiate transcription

E. coli promoters

What promoter is strongest in E. coli?

Consensus 070 dependent promoter

True or False: The more a promoter sequence deviates from the consensus sequence, the stronger it will be

False

True or False: The more a promoter sequence deviates from the consensus sequence, the weaker it will be

True

True or False: The genes belonging to an operon will each have different promoters

False, same promoter

How would you regulate transcription of the Lac operon responsible for lactose catabolism/breakdown?

Use the following:

1) LacI Repressor

2) LacY

1) Represses transcription from Plac (lac operon)

2) Imports lactose into cell

How would you regulate transcription of the Lac operon responsible for lactose catabolism/breakdown?

Use the following:

1) LacZ

2) LacA

1) Hydrolyses lactose → galactose + glucose → energy

2) Unknown function, not required for lactose catabolism

What will happen to the transcription of lactose catabolism/breakdown genes in this situation?

-Low levels of lactose in medium

-Reduce lactose catabolism gene transcription

-LacI repressor binds operator sequence

-Prevents RNA polymerase from transcribing genes

How does LacI repressor prevent RNApol from transcribing genes during low levels of lactose expression?

-Binds operator sequence

-Between promoter and lacZ gene

What will happen to the transcription of lactose catabolism/breakdown genes in this situation?

High

-Induce catabolism genes transcription → energy

-Allolactose isomer binds LacI repressor, stops it

-RNAPol transcribes Plac operon

-IPTG de-represses lacZYA genes transcription

How does allolactose (a natural isomer of lactose) encourage lactose catabolism gene transcription during high levels of lactose expression?

-Binds LacI repressor so it canot bind operator sequence

-RNAPol transcribes from Plac operon

How does IPTG (Isopropyl B-D-1-thiogalactopyranoside) encourage lactose catabolism gene transcription during high levels of lactose expression?

-Synthetic structural analogue to allolactose

-Able to de-repress lacZYA gene transcription

Name one example of Lac repression systems being adapted to expression vectors

Vectors carrying Prac and Ptrc promoters often have lac operator and LacI repressors (lacI, laclq)

What are a characteristic of expression vector pTrc99A?

-Strong transcriptional terminators downstream of cloning site

-rrnB T1 and rrnB T2 avoid destabilizing plasmid during high level transcription

What are these strong transcriptional terminators found in expression vector pTrc99A?

-Avoid destabilizing plasmid during high level transcription

rrnB T1 and rrnB T2

What is fusion vector is this?

-Used for synthesis of fusion proteins

-Recombinant fusion proteins are fused with Maltose-Binding Protein (MBP)

-Improves protein solubility and folding in E. coli

pMal-C2

What is this?

-Combining the coding sequences of two or more genes so they are both transcribed and translated into a single, larger polypeptide chain

-Also known as protein or gene fusion

Translational fusion

What are some characteristics of fusion vector pMal-C2 that make it similar to other expression vectors?

-Ptac promoter

-lacl repressor

-Transcriptional terminators

What must happen before pMal-C2 undergoes translational fusion with MalE?

Gene is cloned

What N-terminal partner protein does pMal-C2 undergo translational fusion with?

MalE (Maltose Binding Protein)

What happens after pMal-C2 fusion vector clones its gene and undergoes translational fusing with MalE?

-Affinity with amylose resin

-MalE can be released from the fusion protein via digestion with Factor Хa

What is this?

-Factor Xa

-Protease that digests/cleaves peptide backbone

-Used to break apart MalE and fusion protein

What is this?

-Affinity for amylose resin/multimers of maltose

-Retained on column after other proteins are washed off during fusion protein purification

MBP (Maltose-Binding Protein)

What happens when you purify and wash a column with MalE-fusion protein?

-Binds to amylose

-Remains on column after washing

What would you use to elute/competitively displace MalE-fusion protein from the column?

Maltose

What is this region used during fusion protein purification?

-Amino acid motif that gets recognized

-Ile Glu Gly Arg

Factor Xa protease cleavage site

What happens when Factor Xa protease recognizes the cleavage site?

-Fusion/recombinant protein is separated from MBР (MalE)

-Can capture MBP on the column and collect released protein in the eluate

Which fusion vector is this?

-Ptac promoter

-lacIq repressor

-N-terminal fusion partner: glutathione-S-transferase

pGEX

pGEX is fusion partners with glutathione-S-transferase.

1) Name the affinity chromatography medium

2) Name what elution occurs with

3) Name the enzyme that cleaves the fusion partner

1) Glutathione-agarose

2) Elution with glutathione

3) 3C protease found in Rhinovirus

Which fusion vector is this?

-PT7 promoter from bacteriophage T7

-No known repressor

-N-terminal hexahistidine fusion partner: Hexahistidine

pRSET

pRSET is fusion partners with Hexahistidine (6xHis)

1) Name the affinity chromatography medium

2) Name what elution occurs with

3) Name the enzyme that cleaves the fusion partner

1) Nickel-sepharose

2) Elution with imidazole (structural analogue of histidine)

3) Enterokinase (EK)

What separation technique is this?

-Mixture of acrylamide/bis-acrylamide (ratio of 29:1)

-Polymerisation initiated with catalysts → porous gel

Polyacrylamide Gel Electrophoresis (PAGE)

What catalysts are used during SDS-PAGE?

Ammonium persulphate + tetramethylethylenediamine

What type of SDS PAGE gel is this?

-4% acrylamide, very porous (on top)

-Concentrate proteins in sample during electrophoresis at the stacking/separating interface

Stacking gel

What type of SDS PAGE gel is this?

-8% - 15% acrylamide (on the bottom)

-Determines pore size of the gel matrix

-Separation of proteins according to mass

Separating gel

How do you prepare a sample for SDS-PAGE?

-Boil in loading buffer

-SDS and reducing agent

What is this?

-SDS (Sodium dodecyl sulphate)

-Strong anionic detergent, denaturing agent

-Denatures proteins

-Associates with protein → Neg (-) charge

Name two examples of reducing agents combined with SDS to make SDS-PAGE loading buffer

2-mercaptoethanol

Dithiothreitol

What is this?

-Reducing agent of SDS PAGE

-Intermolecular disulfide bonds broken to separate subunits

-Internal disulfide bonds broken to denature protein completely

Mercaptoethanol

Where are disulfide bonds formed before they are broken by reducing agents like Mercaptoethanol for SDS-PAGE?

-Between thiol groups

-Found on cysteine amino acids

True or False: Both the gel and electrophoresis both maintain a negative charge on proteins due to SDS content

True

True or False: During SDS, the charge:mass ratio will fluctuate as proteins migrate and separate based on their mass

False, charge:mass ratio remains constant

What is a sign that SDS-PAGE protein denaturation is complete?

-Proper migration

-Accurately reflects mass/size

-Good resolution or separation of proteins

What are the advantages of using denaturing gels for SDS PAGE?

-Predictable migration of proteins on gel

-High resolution separation technique

What are the disadvantages of using denaturing gels for SDS PAGE?

-Cannot study natural protein-protein interactions since complexes are dissociated

-Cannot study natural activities eg. enzymatic activity, receptor binding

What separation technique is this?

-No reducing agents/detergents/no boiling

-Proteins are in native state

-Different shapes, charges

-Migrate according to size, shape and charge

Native PAGE

True or False: Proteins in their native state will all look alike

False, they will have different shapes and charges

What are the advantages of Native PAGE (no denaturing SDS)?

-Can study protein-protein interactions (structures are maintained)

-Can see pilin-pilin and protein-chaperone interactions

-Can study enzyme activities (zymogram), binding activities with other molecules

What are the disadvantages of Native PAGE (no denaturing SDS)?

-Protein migration unpredictable

-Mass determination not possible

How can you detect proteins in SDS and native gels?

-Coomassie Blue staining

-Silver staining (more sensitive)

-Sypro Ruby, other sensitive dyes

What is this protein detection technique?

-Identify specific protein expression in a complex mixture (100s to 1000s of proteins)

Western Blot

How does Western Blot work?

1) Electrophoretic transfer of proteins from PAGE gel to a membrane

2) Block non-specific protein binding sites on membrane by incubation with protein

3) Incubate membrane with antibody specific for protein of interest

4) Detect binding of primary antibody with a secondary antibody-enzyme conjugate

5) Detection based on enzyme activity of secondary antibody conjugate

What is the first step of Western Blot?

Electrophoretic transfer of proteins from PAGE gel to a membrane

True or False: Western Blot only works with SDS denaturing gel

False, it works with both denaturing and native gel

What membrane can proteins be electrophoretically transferred to via Western Blot?

-Nitrocellulose

-PVDF

What happens after electrophoretic transfer of proteins from PAGE gel to a membrane during Western Blot?

Block non-specific protein binding sites on membrane by incubation with protein

What protein can you use to block non-specific proteins from binding to the membrane during Western Blot?

-Bovine Serum Albumin

-Tween 20 (mild non-ionic detergent)

Why do we block non-specific protein binding sites on the membrane during Western Blot?

-Stops antibodies binding non-specifically to the membrane and other proteins

-Reduces background reactivity

What happens after non-specific protein binding spites on the membrane are blocked during Western Blot?

Incubate membrane with 1st antibody that is specific to protein of interest

What would you use this for during Western Blot?

-Rabbit anti-MalE antibody

Incubate membrane with 1st antibody that is specific to protein of interest

What happens after incubating the membrane with an antibody specific to the protein of interest during Western Blot?

Detect binding of primary antibody with secondary antibody-enzyme conjugate

What would you use this for during Western Blot?

-Goat anti-rabbit IgG-alkaline phosphatase conjugate

Detect binding of primary antibody with secondary antibody-enzyme conjugate

What happens after detecting the binding of a primary antibody to a secondary antibody-enzyme conjugate?

Detection based on enzyme activity of secondary antibody conjugate