Mirco Lab Midterm: Lecture 3

Question 1

Describe the purpose of recombinant proteins. What are they used for?

Answer 1

• Analysis of activity
• Study of structure-function relationship
• Make antibodies
• Produce enzymes for biotechnology
• Produce proteins for application in medicine
• Produce vaccines

Question 2

How expression systems are selected?

Answer 2

• Size
• Proteolitic cleavage (only mammalian and insect systems)
• Purity/yield: E.coli(>50%), mammalian (<1%), insect (>30%),
  yeast (1%)
• Amounts needed: functional studies (μg); antibody production
  (mg); biotech (g-kg)
• Native (active)/denatured (epitopes exposed)
• Post-translationally modified/not modified
• Sequence: codon preference
• Cost/speed
• Availability of the system/investment to test new system

Question 3

What are the steps of making recombinant proteins?

Answer 3

Clone or synthesize the ORF of the protein
* Clone in expression vector
* Transform (transfect) cells
* Grow the cells and induce the expression
* Purify the protein
* Warning: Expression and purification of
recombinant proteins is not trivial

Question 4

What are the protein expression requirements?

Answer 4

Presence of a strong promoter

• Efficient translation
• Posttranscriptional modifications: needed or
  not
• Stability of the protein

Question 5

What are promoters?

Answer 5

Promoters are sequences to which RNA pol
binds and initiates transcription

• E. coli RNA pol or T7 phage RNA pol are
  employed by all expression systems in E. coli

Question 6

In this case, what is translation?

Answer 6

Translation in E. coli depends on the presence of ShineDelgarno sequence: GGAGG

• S-D sequence is complementary to 3’ end of 16s
  ribosomal RNA
• Translation efficiency depends on:
• degree of complementarity of S-D with 16s RNA,
• distance between the promoter and S-D,
• +1 should be more than 15bp and
  about 50bp from first codon AUG,
• S-D sequence should be less than 10bp from the AUG, +1 and S-D must be
  accessible (not folded in secondary structure)

Question 7

In this case, what is the codon choice(s)

Answer 7

• The synonymous codons are not used with
  equal frequency
• Correlation with tRNA availability
• The first 7-8 codons of the expressed proteins
  can dramatically change the expression of the
  recombinant protein – rare codons like AGA
  and AGG, high GC content decrease the
  expression