Recombinant Proteins Flashcards
(35 cards)
What organism is GFP originally derived from?
Aequorea victoria, a jellyfish.
What is GFP used for in cell biology?
Tracking protein expression, localization, protein-protein interactions, and live cell imaging.
What modifications have been made to recombinant GFP?
Increased brightness, photostability, and altered color variants (e.g., CFP, YFP, RFP).
How is GFP typically inserted into a gene construct?
Through in-frame fusion to the gene of interest using restriction sites or PCR.
What ensures high expression of GFP fusion proteins in mammalian systems?
Strong promoters like CMV.
What are the two main GFP cloning strategies?
Restriction digest-based and PCR-based strategies.
When is restriction digest-based cloning preferred?
When a large amount of plasmid and compatible MCS are available.
What is required in restriction digest cloning?
Identical restriction sites on vector and insert, followed by ligation.
When is PCR-based cloning used?
Recomb proteins
When plasmid amounts are limited or when generating modified constructs.
How does PCR-based cloning enhance flexibility?
Allows rapid generation of variants and site-specific modifications.
What are the common types of GFP fusions?
C-terminal (Protein–GFP), N-terminal (GFP–Protein), and internal insertions.
Choosing the right fusion type ensures your GFP-tagged protein behaves like the native one
What vector element ensures protein expression in mammalian cells?
The pCMV promoter.
Think of the pCMV promoter as a green light or “on switch” in a DNA plasmid. It tells mammalian cells:
“Hey, start making this protein from this DNA!”
It’s like the engine starter in a car—without it, the car (cell) won’t produce the protein.
What vector element stabilizes mRNA transcripts?
BGH polyadenylation signal.
Once a gene is read and turned into mRNA, you want that mRNA to stay stable long enough to make the protein. The BGH polyA signal is like tying a knot at the end of a rope—it protects the mRNA from getting cut up too quickly in the cell.
What allows selection in mammalian systems?
Neomycin resistance gene.
What allows for plasmid propagation in bacteria?
Ampicillin resistance and pUC origin.
Ampicillin resistance gene: Helps you select only the bacteria that got the plasmid—others die in the antibiotic.
pUC origin: This is the part of the plasmid that tells bacteria how to copy it properly—like printing instructions.
Name two GFP-like proteins from Anemonia sulcata and their colors.
asFP499 (green) and asFP595 (red).
Why are non-traditional GFP-like proteins useful?
Enable multi-color imaging in live cells.
What is eqFP611 and its source?
A red fluorescent protein from Entacmaea quadricolor.
What are eqFP611’s advantages in imaging?
Deeper tissue penetration, reduced autofluorescence, and dual-color compatibility.
How is eqFP611 used in functional studies?
As a fluorescent tag for proteins like the Notch receptor in mammalian cells.
What can GFP fusion interfere with?
Protein function due to steric hindrance or misfolding.
How was Notch interference detected in the study?
Via a luciferase reporter assay showing functional impairment.
Notch interference refers to a disruption in the Notch signaling pathway, which is important for cell communication and development.
A luciferase reporter assay is a lab technique used to measure gene expression. Scientists attach a luciferase gene (which makes cells glow) to a DNA sequence controlled by the Notch pathway.
If Notch is working properly, the luciferase gene will be expressed, and cells will glow.
So, the study detected Notch interference by seeing a reduced glow in cells—indicating impaired Notch activity.
What tag is used in IMAC purification?
A 6×His-tag.
How does IMAC work?
His-tag binds to Ni²⁺/Co²⁺ resins; eluted by imidazole or pH shift.
