Ch. 10 Recombinant Proteins Flashcards
Which of the following is a clinically relevant protein produced by recombinant technology?
All of the above
- insulin
- interferon
- factor VIII
- tissue plasminogen activator
Which of the following is associated with translation efficiency for the mRNA of genes cloned for protein expression?
All of the above
- mRNA stability
- strength of the ribosomal binding site
- codon usage
- mRNA secondary structure
Which of the following is NOT a feature of translational expression vectors?
The native promoter for the gene that is to be cloned into the vector
Which method to overcome codon usage problems is the least labor-intensive?
Supply the genes for the rare tRNAs on a separate plasmid
In expression systems, which of the following forms when too much protein is manufactured too quickly?
T7 RNA polymerase
How can recombinant protein production be enhanced?
Co-expression of a molecular chaperone
What can be done to ensure a recombinant protein is excreted outside the cell?
All of the above
- addition of a signal sequences
- using specialized export systems
- fusion to a protein that is normally exported
- expressing the protein in an alternative system, such as Gram-positive bacteria
What could be used to increase the stability and purification of eukaryotic proteins from bacterial cells?
A peptide tag
Why would it be necessary to express eukaryotic proteins in eukaryotic cells rather than in bacterial cells?
Protein production in eukaryotic cells never produces inclusion bodies, which sometimes form in bacterial cells due to overexpression
Which of the following is not a problem associated with low yields of proteins expressed in yeast?
Proteins are not getting secreted into the medium
What is a bacmid?
A shuttle vector that replicates as a plasmid in E. coli and a virus in insect cells
Are insect cells fully able to glycosylate foreign proteins?
No
Which of the following is not used as a selectable marker to maintain mammalian shuttle vectors within mammalian cells?
Glutamine synthetase gene
What function does an internal ribosomal entry site serve?
As a protease cleavage site in between two proteins expressed in one transcript
Which factor affects which protein expression system to utilize?
All of the above
- cost
- ability to glycosylate or fold proteins
- speed
- government regulations
Which of the following is not involved in the manufacturing of insulin?
Phosphodiesterase 5
Which of the following statements about insulin manufacturing is NOTes true?
Bacteria are able to express preproinsulin and process this peptide into insulin
The recently discovered autotransporter proteins of bacteria:
Require the BAM complex to cross the bacterial outer membrane
True about insulin manufacturing
- diabetics used to have to inject themselves with insulin that had been purified from the pancreas of either cows or pigs
- natural insulin forms hexagamers, which causes the hormone to clump
- a proline converted to an aspartic acid keeps insulin from clumping
- recombinant insulin is expressed as two mini-genes
Features of translational expression vectors
- a selectable marker
- terminator sequences
- consensus RBS plus the ATG initiation codon
- a strong, but regulated, transcriptional promoter
Inclusion bodies
dense crystals if misfolded and nonfunctional protein
Joining the coding sequences of two proteins together in frame makes a ___ ___.
Protein fusion
Type I secretory system -
Spans both inner and outer membranes and is used to secrete hemolysin by some E. coli strains that cause urinary infections
Type II secretory system -
Spans the outer membrane only
