GST, GFPS65T, and a GST-GFPS65T fusion proteins. 1. What is the purpose of the a
ID: 225713 • Letter: G
Question
GST, GFPS65T, and a GST-GFPS65T fusion proteins.
1. What is the purpose of the ampicillin in the bacterial growth media?
2. What does PMSF do? Why would you include it in a purification?
3. What is the difference between the protein profile of induced and non-induced cells?
4. Check your calculations for the molecular weight of GST, GFPS65T, and a GST-GFPS65T fusion protein (assume residues LVPRGS in the linker between GST and GFP).
5. Compare your pI (isolectric point) calculations for GST, GFPS65T, and a GST-GFPS65T fusion protein (assume residues LVPRGS in the linker between GST and GFP). Is the pI important when you are purifying a protein? Why? What would happen if the pH of the buffer was the same as the pI?
6. What does a hydropathy plot of GST, GFPS65T, and a GST-GFPS65T fusion protein show? Do you think that the hydropathy plot profile of a protein has any relevance to how you would purify the protein?
1. What is the purpose of the ampicillin in the bacterial growth media? 2. What does PMSF do? Why would you include it in a purification? 3. What is the difference between the protein profile of induced and non-induced cells? 4. Check your calculations for the molecular weight of GST, GFPS65T, and a GST-GFPS65T fusion protein (assume residues LVPRGS in the linker between GST and GFP). 5. Compare your pl (isolectric point) calculations for GST, GFPS65T, and a GST-GFPS65T fusion protein (assume residues LVPRGS in the linker between GST and GFP). Is the pl important when you are purifying a protein? Why? What would happen ifthe pH of the buffer was the same as the pl? 6. What does a hydropathy plot of GST, GFPS65T, and a GST-GFPS65T fusion protein show? Do you think that the hydropathy plot profile of a protein has any relevance to how you would purify the protein?Explanation / Answer
This question has multiple questions and the asker did not which question to be answered. As per Chegg's policy, I am answering the first 4 questions.
1) Ampicilin is added to bacterial growth media to select transformants after cloning. If you clone a gene in a plasmid containing beta-lactamase gene (Ampicilin resistance gene), and transform bacteria with that plasmid, bacterial cell can grow on media containing Ampicilin. Bacterial cell that did not plasmid will not have ampicilin resistance, and cannot survive.
2) PMSF (phenylmethylsulfonyl fluoride) is a a serine protease inhibitor. It will be added to cell lysates to prevent the activity of proteases in the cell lysates. All cells may have some proteolytic enzymes at any given point time. When the cells are lysed, these proteases are free to react and degrade other protein. To prevent this. PMSF will be added to cell lysates before/during protein purification.
3) Cell lysate of recombinant clones after and before induction of recombinant gene expression along with protein molecular weight size standard can be loaded on SDS-PAGE gel and check the protein expression by coomassie or silver staining. This is called protein profile. We will see high level of expression of recombinant protein after induction as band corresponding to the molecular weight of recombinant protein. We will not see this band in non-induced cell lysate.
4) Molecular weights
GST= 25.499 KD GFPS65T= 26.928 KD GSTGFPS65T= 53.019
Protein sequence of the above three proteins can be found in the end of the answer.
5) pI
GST= 6.09 GFPS65T= 5.8 GSTGFPS65T= 6
If pH of buffer is same as pI of protein, net charge of protein in that buffer is zero.
So, pI is important when we are purifying protein. If we are using a purification technique (eg. Ion exchange chromatography) that separates based on charge on protein, we should consider pI of protein and pH of buffer.
Protein sequences:
gst (218 aminoacids)
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK
gfps65t (238 aminoacids)
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK
gst-gfps65t with LVPRGS linker (218+6+238 aminoacids)
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK
LVPRGS
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK
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