Lecture 3 Flashcards
(31 cards)
What percentage of the initial material can a target protein represent?
Less than 0.1% by weight of initial material.
What are the four main requirements for purifying a protein?
Need a source of the protein.
* Need a means of isolating (or extracting) the protein from the source.
* Need to be able to detect (measure) the protein.
* Need to be able to stabilize the protein.
What are the two main sources of proteins for purification?
Natural (Endogenous) and Recombinant (Heterologous).
Briefly describe how a recombinant protein is made.
The gene for the protein is inserted into a host organism using a vector, and then its expression is induced in the host.
What are the three key steps in Recombinant DNA Technology (Cloning) for protein expression?
- Polymerase Chain Reaction (PCR), 2. Transformation, 3. Expression.
Compare E. coli (prokaryotic host) and Baculovirus-infected insect cells (eukaryotic host) regarding post-translational modifications.
- E. coli: Proteins synthesized without post-translational modification, no eukaryotic chaperones.
- Insect cells: Eukaryotic proteins synthesized with proper post-translational modifications, chaperones present.
List three advantages of using bacterial cells for protein expression.
Fast growth, affordable medium, affordable equipment, well-studied expression hosts.
What are two main limitations of bacterial cells for expressing eukaryotic proteins?
Different codon usage frequencies (leading to low yield for some codons).
* Lack of eukaryotic post-translational modifications (e.g., disulfide bonds, glycosylation).
What is the solution for codon usage differences when expressing eukaryotic proteins in bacteria?
Synthesize a codon-optimized version of the gene.
What is the solution for lack of post-translational modifications when expressing eukaryotic proteins in bacteria?
May have to use eukaryotic cells.
What are the three goals when expressing a protein?
Obtain as much product as possible, obtain soluble and correctly folded protein, and obtain it in a form that is easy to purify.
How is high protein expression a problem, and what is a solution?
High expression can lead to aggregation and decreased cell growth. Solution: need inducible expression.
How can you make separating your expressed protein from E. coli proteins easier?
Need a “tag” on the expressed protein to facilitate separation.
What is the function of a Promoter?
Regulatory region upstream of a gene that controls transcription by providing binding sites for RNA polymerase and other factors.
What is an Operator?
A special DNA sequence between a promoter and structural genes (e.g., in the lac operon) that allows repression of gene expression when an inhibitor protein binds to it.
What is a Ribosome Binding Site (RBS)?
An RNA sequence on mRNA to which ribosomes bind to initiate translation. In bacteria, it’s almost always required along with a start codon.
What is the function of Terminators?
Genetic parts at the end of a gene or operon that cause transcription to stop.
What is a Stop Codon?
A three-nucleotide sequence in DNA or mRNA that signals a halt to protein synthesis
What is a Gene?
Information encoded in DNA to be expressed into a function, usually via RNA coding for proteins or non-coding RNA.
What is the Start Codon and what amino acid does it code for?
ATG, which codes for methionine.
What is the Origin of Replication (ORI) and why is it often A-T rich?
The place where DNA replication begins on a plasmid, enabling it to reproduce. It’s A-T rich because A-T pairs have two hydrogen bonds (vs. three in G-C), making them easier to separate for replication.
List three common stop codons (DNA or mRNA).
TAG (UAG), TGA (UGA), TAA (UAA).
List five factors important for protein stabilization
pH (buffering), temperature (typically 4°C), inhibiting degradative enzymes, preventing adsorption to surfaces, and proper long-term storage (-80°C, inert gas).
What are the three characteristics of a good assay?
Specific, Sensitive, and Convenient.
