Let’s say you use hypotonic swelling and needle-shearing to lyse millions of hum
ID: 65078 • Letter: L
Question
Let’s say you use hypotonic swelling and needle-shearing to lyse millions of human liver cells, and you place this lysate in a test tube already containing isoprenylated cysteines (which are a waste product that rapidly accumulates in cells when Ras and other isoprenylated proteins are degraded). You detect that the isoprenoid lipid is rapidly removed from the cysteine in the test tube, and you decide to name the unpurified cellular molecule that is catalyzing this reaction prenylcysteine lyase (PCLase). Write a detailed and thorough outline of how you would: 1) ascertain that PCLase is actually a protein and not some other catalytic molecule like RNA, 2) purify PCLase (several approaches might be considered for this; describe them), 3) locate a human chromosomal region (not a cDNA) that harbors an exon of the gene encoding PCLase (recall that the human genome is now fully sequenced and available), 4) clone (physically isolate) the entire human gene (not the cDNA) that encodes PCLase, and 5) verify that the gene you cloned indeed encodes a protein with the catalytic properties of PCLase. Please include all important steps of this process.
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Explanation / Answer
1. Every protein has caharacteristic one CO terminus, peptide bonds and one NH2 terminus. It can be recognized.
2. Purification of PCLase can be either based on the functional properties of the protein, affinity to certiain ligand, or sedimentation at certain pH or it can be cloned and overexpressed in an invitro cell model using expression vectors
3. PCLase gene sequence can be blasted and exon can be found out by comparing it with the mRNA sequence
4.Cloning of PCLase can be done using either cloning vectors or expression vectors. using restriction enzymes, transformation and selection
5.Functional properties of PCLase can be varified using the same functional properties which were used during purification of the protein
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