otein biomarker for breast cancer has 120 amino acids, single chain. I want a fi
ID: 693593 • Letter: O
Question
otein biomarker for breast cancer has 120 amino acids, single chain. I want a fiuoresce e uch as coumarin at its #6 amino acid position for cancer staining surgery. List 4 possible rooaches you can think of to engineer 1 g such protein. Please recommend with compar aanation which approach is the best one to be used in hospital in terms of purity and activity eeip if you can use some drawings (multiple points, points will be deducted if you choose t e recommend with comparison and urity and activity. It will ts will be deducted if you choose the wrong approaches) (10 points) Fluorescent probe: CoumarinExplanation / Answer
The four possible approaches are
1) Genetically encoded labels- The Aequorea victoria is a Green fluorescent protein (GFP). It was first used as a fluorescent marker. The fluroscent protein fused with the target protein at the N or C terminus. It is easily done through recombinant clonining.
2) Direct labeling with organic fluorophores- In this direct chemical labelling is done to purified protein that targets cystein and amines groups. The introduction of cysteines at specific site is done for labelling using site directed mutagenesis. This is done to convert the surface exposed to cystein. The greatest flexiblity is allowed by cystein as it allows to choose location for labelling due to low flexiblity. Complications that arrises with labeling cysteines is because of the presence of multiple target residues.
3) Peptide tags for organic fluorophores-There are two approaches for this- the target protein is attached to a synthetic peptide through ligation or genetic engineer peptides tags on the target protein which then react with the flurophore conjugate. Another one is C-terminal labelling- In this the target protein has to be produced with a C-terminal thioester
4) Protein tags for organic fluorophores- The protein tags such as Halo, SNAP, and CLIP, these are self-labeling enzymes as they covalently link to fluorescently labeled substrates. They are highly specific. For labelling the protein of interest O6-benzylguanine substrates are used.
In this the genetically encoded labels is the good one, as it is the most specific approach for labelling proteins that does not result in the background signal from free flurophore.
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