L4: Production of Proteins using E. coli, mammalian and insect cells

Question 1

Disadvantage of using E. coli as a host cell

Answer 1

Can lead to inclusion bodies

Question 2

Translation cassette elements

Answer 2

ATG start codon
Promoter for use in prokaryotes
Shine-Dalgarno - RBS in mRNA of prokaryotes, located ~16 nt upstream of the start codon GAAGGAG 
Distance between the SD and the ATG
Strength of RBS including ATG

Promoter for use in Eukaryotes
Kozak sequence for initiation of translation on eukaryotic mRNA  gccRccAUGG  R=A or G   
TATA box (25-30 nt upstream of start in some eukaryotic promoters) binding protein (TBP) transcription factor that binds to the TATA box as part of the pre-initiation complex and helps position the RNA polymerase II over the start of the gene
May have Internal Ribosome Entry Site (IRES before a second gene)

Downstream of gene to be translated
Nucleotide Stop codon (hard stop TAA, soft stop TAG (amber read-through)
Protein Transcription Terminators (CCAAT-box)
Polyadenylation site in eukaryotes

Question 3

Advantages and disadvantages of E. coli as expression symptoms

Answer 3

Disadvantages: pyrogens (lipopolysaccharide), lack of eukaryotic protein modifications, inclusion bodies, max protein size ~100kDa

Advantages: Simple, inexpensive, high level protein production, inclusion bodies can sometimes be used to an advantage.

Question 4

Why use yeast for the production of valuable proteins?

Answer 4

Eukaryote
Transformation
Non-pathogenic
Growth and culture are inexpensive and easy
Genetics and molecular biology is well understood
Reasonable track record
Possible secretion of protein
SAFE