Let’s say you use hypotonic swelling and needle-shearing to lyse millions of hum
ID: 65589 • 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. For full credit, your
outline must include all important steps of this process.
Explanation / Answer
1. To ascertain the new cellular molecule as protein can b differentiated form nucleic acids by comparing the absorbance at 260nm and at 280nm. Protein will show maximum absorbance at 280nm. Also positive biuret test will differentiate proteins from nucleic acids.
2. Purification of PCLAse can done using methods like ion exchange, affinity chromatography. The proteins are usually purified on the basis of size and charge. More discriminating separations on the basis of size can be achieved by the technique of gel-filtration chromatography. The crude lysate cotaining PCLase will applied onto the column, the larger proteins will elute first and at the last the smaller proteins elute. Proteins can be separated on the basis of their net charge by ion-exchange chromatography. If a protein has a net positive charge at pH 7, it will usually bind to a column of beads containing carboxylate groups, whereas a negatively charged protein will not. Affinity chromatography is another powerful and generally applicable means of purifying proteins. This technique takes advantage of the high affinity of many proteins for specific chemical groups.
3. The location of PCLase exon on the chromosome can be using FISH (Fluorescent in situ hybridisation) technique. FISH uses a fluorescently labelled hybridisation probe which will attach to the exact part of the DNA which codes for the protein. The hybridisation probe is a short fragment of DNA that has a fluorescent dye attached. A typical FISH probe would be 10 - 100 kb. The probe is added to a microscope slide where all the chromosomes from a cell are spread out and have been denatured (treated so that their DNA is single-stranded).
4. The clone of the entire region of the PCLase gene can be done using the primers for the gene created using the gene as a template. Then the gene will amplified from the genome using the forward and reverse primers, nucleotides and Taq DNA polymerase in a eppendorf tube. The amplified gene product will be isolated, purified and digested with restriction enzymes and ligated with a bacterial vector. To confirm the ligation, the vector will transformed into a competent E.coli cells and plated on the agar plate with a antibiotic as a resistance marker.
5. To verify the cloned gene encodes the protein, you need tp express the cloned gene in a expression vector and transform into the bacterial cells. Isolate the total bacterial lysate and purify the PCLase to near homogenity using a his tag affinity purification which is present in the vector and test the enzyme activity to confirm the intact nature of the protein.
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