Protein Production Methods

Question 1

How can we produce large amounts of protein

Answer 1

Recombinant Protein Expression

Question 2

Overview of cloning methods

Answer 2

• Inserting a fragment of DNA (the gene encoding your target protein) into a vehicle (plasmid) for its replication within a host organism
• Sources of template dna (genomic dna, cDNA, plasmids, synthetic genes etc)
• Once you have some starting material, the first step is to amplify that DNA by PCR

Question 3

PCR Overview

Answer 3

Denaturation (95-98) - Separates double stranded DNA into 2 single strands by breaking H-bonds creating accessible templates for primers

Annealing (50-60) - Temp lowered to allow primers to bind to complementary sequences on single stranded DNA. Specificity ensures that only the desired DNA segment is amplified.

Extension (72) - DNA polymerase synthesizes new DNA strands by adding nucleotides to the bound primers.

Question 4

Traditional vs Modern

Answer 4

Question 5

Traditional Cloning

Answer 5

Question 6

Modern Cloning

Answer 6

Question 7

Seamless Cloning: In fusion

Answer 7

• Ligation-free insertion of PCR products into any vector
• Cloning reaction relies on 15-20bp overlapping homology between DNA fragment ends => ensures that reaction is directional and accurate
• Infusion enzyme utilizes 3’-5’ exonuclease activity to remove nucleotides from the 3’ end exposing
  complementary regions on the insert and vector which can be annealed together spontaneously
• Joining of DNA fragments can be facilitated by a recombination event directed by short att recombination sites flanking the linearised vector and PCR product insert
• target gene can be cloned directly into the final expression vector, without the need of first cloning into
  an entry vector as for Gateway cloning.

Question 8

Advantages of Seamless cloning

Answer 8

Question 9

Choosing a heterologous expression host

Answer 9

Question 10

Choosing a plasmid (replicon, multiple cloning site, antibiotic resistance gene, promoter)

Answer 10

Replicon
- Origin of DNA replication
- Determines plasmid copy number

Multiple Cloning Site
- Sequence downstream of promoter which contains multiple restriction sites allowing for easy insertion of DNA

Antibiotic Resistance Gene
- Allows for selection of plasmid containing bacteria

Promoter
- Drives transcription of target gene
- Determines which cell types the gene is expressed in and the amount of recombinant protein obtainedCh

Question 11

Choosing a plasmid (Affinity tag)

Answer 11

Question 12

Choosing a plasmid (T7 lac systems)

Answer 12

Question 13

Standard Expression Method

Answer 13

Question 14

Induction

Answer 14

Question 15

High throughput cloning & expression

Answer 15

Question 16

Optimization & Troubleshooting

Answer 16

Question 17

Choosing an expression system

Answer 17

Bacmids

Mammalian Cells
- More fragile so more demanding culture conditions
- High level of protein processing
- Most common cell lines for protein expression