Homework Help is Here – Start Your Trial Now!
Science
Biochemistry
proOT NP I J Processing V Eneymes Ž:3 OT You have been tasked to purify mature OT from a cell lysate expressing proOT-NPI that has been tagged with a polyhistidine tag at the C-terminus. Design a purification scheme to accomplish this goal. You have all of the biochemical equipment we've discussed in class, as well as purified Processing Enzymes 1, 2, and 3. The MW and pl values for proOT-NPI, NPI, proOT, and OT are provided in the table to the right. You should assume conversion reactions will not go to completion, so you will need to purify any products from the starting material. Assume that compounds must be 1000 Da different to separate with size- exclusion chromatrography and have a pl difference of 1 to separate via ion exchange chromatography. Your answer can include a drawing, but include some text so that I can understand your rationale for each step. Protein MW (Da) pl ProOT-NPI 10593 4.94 NPI 9600 4.94 ProOT 1010 5.51 от 1007 N/A* *OT is modified such that it neutral except for one ionizable group, which is the phenolic -OH of tyrosine (pK=10).

proOT NP I J Processing V Eneymes Ž:3 OT You have been tasked to purify mature OT from a cell lysate expressing proOT-NPI that has been tagged with a polyhistidine tag at the C-terminus. Design a purification scheme to accomplish this goal. You have all of the biochemical equipment we've discussed in class, as well as purified Processing Enzymes 1, 2, and 3. The MW and pl values for proOT-NPI, NPI, proOT, and OT are provided in the table to the right. You should assume conversion reactions will not go to completion, so you will need to purify any products from the starting material. Assume that compounds must be 1000 Da different to separate with size- exclusion chromatrography and have a pl difference of 1 to separate via ion exchange chromatography. Your answer can include a drawing, but include some text so that I can understand your rationale for each step. Protein MW (Da) pl ProOT-NPI 10593 4.94 NPI 9600 4.94 ProOT 1010 5.51 от 1007 N/A* *OT is modified such that it neutral except for one ionizable group, which is the phenolic -OH of tyrosine (pK=10).

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
See similar textbooks
icon
Related questions
Question
Please help. An explanation will be very helpful. Thank you
**Title: Understanding the Biochemical Processing of Oxytocin** **Introduction:** Oxytocin is a 9-residue peptide hormone involved in signaling pathways related to reproduction and perinatal health. Initially produced as a proprotein, oxytocin is synthesized as Oxytocin-neurophysin I (proOT-NPI), which is further processed into functional proteins. **Diagram Overview:** 1. **Initial Processing:** - ProOT-NPI undergoes processing via **Enzyme 1** to split into proOT and neurophysin I (NPI). - The initial structure includes a polypeptide chain extended by a polyhistidine tag (6xHis) at the C-terminus. 2. **Further Processing:** - ProOT undergoes further processing using **Enzymes 2 and 3** to yield active oxytocin (OT). - This step involves the removal of a Lys-Arg dipeptide. **Purification Task:** You are tasked with purifying mature OT from a cell lysate expressing proOT-NPI with a C-terminal polyhistidine tag. The molecular weight (MW) and pI values for proOT-NPI, NPI, proOT, and OT are as follows: | Protein | MW (Da) | pI | |-------------|---------|------| | ProOT-NPI | 10593 | 4.94 | | NPI | 9600 | 4.94 | | ProOT | 1010 | 5.51 | | OT | 1007 | N/A* | *Oxytocin is modified to remain neutral except for the ionizable phenolic OH of tyrosine (pKᵃ=10). **Purification Strategy:** - **Assumptions:** - Conversion reactions may be incomplete; purification from starting material may be required. - Compounds must differ by at least 1000 Da for separation using size-exclusion chromatography. - A pI difference of 1 is necessary for ion exchange chromatography. - **Approach:** - Employ biochemical equipment and purified processing enzymes discussed in class. - Design a systematic purification scheme, possibly incorporating a drawing, and explain each step's rationale. Understanding and implementing these strategies will aid in the efficient purification of oxytocin, ensuring its functionality for physiological applications.
Transcribed Image Text:**Title: Understanding the Biochemical Processing of Oxytocin** **Introduction:** Oxytocin is a 9-residue peptide hormone involved in signaling pathways related to reproduction and perinatal health. Initially produced as a proprotein, oxytocin is synthesized as Oxytocin-neurophysin I (proOT-NPI), which is further processed into functional proteins. **Diagram Overview:** 1. **Initial Processing:** - ProOT-NPI undergoes processing via **Enzyme 1** to split into proOT and neurophysin I (NPI). - The initial structure includes a polypeptide chain extended by a polyhistidine tag (6xHis) at the C-terminus. 2. **Further Processing:** - ProOT undergoes further processing using **Enzymes 2 and 3** to yield active oxytocin (OT). - This step involves the removal of a Lys-Arg dipeptide. **Purification Task:** You are tasked with purifying mature OT from a cell lysate expressing proOT-NPI with a C-terminal polyhistidine tag. The molecular weight (MW) and pI values for proOT-NPI, NPI, proOT, and OT are as follows: | Protein | MW (Da) | pI | |-------------|---------|------| | ProOT-NPI | 10593 | 4.94 | | NPI | 9600 | 4.94 | | ProOT | 1010 | 5.51 | | OT | 1007 | N/A* | *Oxytocin is modified to remain neutral except for the ionizable phenolic OH of tyrosine (pKᵃ=10). **Purification Strategy:** - **Assumptions:** - Conversion reactions may be incomplete; purification from starting material may be required. - Compounds must differ by at least 1000 Da for separation using size-exclusion chromatography. - A pI difference of 1 is necessary for ion exchange chromatography. - **Approach:** - Employ biochemical equipment and purified processing enzymes discussed in class. - Design a systematic purification scheme, possibly incorporating a drawing, and explain each step's rationale. Understanding and implementing these strategies will aid in the efficient purification of oxytocin, ensuring its functionality for physiological applications.
Expert Solution
Step 1

PrO-OT-NPI has six Histidine residues in the tag form. Processing enzyme I convert it into Pro-Ot and NPI. There is a large difference between the molecular weights of Pro-OT and NPI. Now by processing enzymes 2 and 3, Pro-OT will be converted to OT or active form of oxytocin. OT is present in the neutral form except at one residue which is phenolic group of tyrosine, it means its PI value is around neutral pH. So, Pro-OT and OT has a difference of more than 1 between their PI values. 

steps

Step by step

Solved in 2 steps with 1 images

SEE SOLUTIONCheck out a sample Q&A here
Blurred answer
Knowledge Booster
Forensic Analysis
Learn more about
Need a deep-dive on the concept behind this application? Look no further. Learn more about this topic, biochemistry and related others by exploring similar questions and additional content below.
Recommended textbooks for you
Biochemistry
Biochemistry
Biochemistry
ISBN:
9781319114671
Author:
Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:
W. H. Freeman
Lehninger Principles of Biochemistry
Lehninger Principles of Biochemistry
Biochemistry
ISBN:
9781464126116
Author:
David L. Nelson, Michael M. Cox
Publisher:
W. H. Freeman
Fundamentals of Biochemistry: Life at the Molecul…
Fundamentals of Biochemistry: Life at the Molecul…
Biochemistry
ISBN:
9781118918401
Author:
Donald Voet, Judith G. Voet, Charlotte W. Pratt
Publisher:
WILEY
Biochemistry
Biochemistry
Biochemistry
ISBN:
9781305961135
Author:
Mary K. Campbell, Shawn O. Farrell, Owen M. McDougal
Publisher:
Cengage Learning
Biochemistry
Biochemistry
Biochemistry
ISBN:
9781305577206
Author:
Reginald H. Garrett, Charles M. Grisham
Publisher:
Cengage Learning
Fundamentals of General, Organic, and Biological …
Fundamentals of General, Organic, and Biological …
Biochemistry
ISBN:
9780134015187
Author:
John E. McMurry, David S. Ballantine, Carl A. Hoeger, Virginia E. Peterson
Publisher:
PEARSON
SEE MORE TEXTBOOKS