1.8 Calculations for the Purification of a Recombinant Protein The purification of arecombinant protein is carried out starting with 100 liters of a clarified cell lysate (i.e., thecells have been lysed, and the cell debris has been removed to give a clarified solution),which has a total protein concentration of 0.36 mg/ml and a recombinant protein con-centration of 2.2 U/ml, where U denotes units of biological activity of the recombinantprotein. It is known that the completely pure recombinant protein has a specific activityof 40.0 U/mg. Purification is continued until a chromatography step that yields 2.0 litersof a fraction containing the protein, with a total protein concentration of 1.11 mg/mland a recombinant protein concentration of 43.2 U/ml.For the recombinant protein,calculate the starting and ending purity, the starting and ending specific activity, and thepercentage yield and fold purification through the chromatography step.

Purification of a Recombinant Protein:This process involves the following steps-Cel harvesting- In…

Answered: starting and ending purity, t

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter29: Transcription And The Regulation Of Gene Expression

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Calculations for the Purification of a Recombi- nant Protein The purification of a recombinant protein is carried out starting with 100 liters of a clarified cell lysate (i.e., the cells have been lysed, and the cell debris has been removed to give a clar- ified solution), which has a total protein concentra- tion of 0.36 mg/ml and a recombinant protein con- centration of 2.2 U/ml, where U denotes units of biological activity of the recombinant protein. It is known that the completely pure recombinant pro- tein has a specific activity of 40.0 U/mg. Purifica- tion is continued until a chromatography step that yields 2.0 liters of a fraction containing the protein, with a total protein concentration of 1.11 mg/ml and a recombinant protein concentration of 43.2 U/ml.. For the recombinant protein, calculate the. starting and ending purity, the starting and ending specific activity, and the percentage yield and fold purification through the chromatography step. :
2.7 Problem in the Determination of Molecular Welght by Gel Electrophoresis A puri- fied recombinant protein is analyzed for molecular weight by SDS-PAGE at pH 8.5. From the protein sequence deduced from the gene that was expressed in bacteria, the protein is expected to have a molecular weight of 44,000. However, the molecular weight of the pro- tein is found by SDS-PAGE to be 52.000. Explain the reason or reasons for this difference in molecular weight. What calculation could you make to help explain this discrepancy?
Determination of MW by Gel electrophoresis
8.
One of the useful rules of thumb that you may have learned in Biochemistry, is that a 50 g/ml solution of DNA has an absorbance of about 1.0 in a 1 cm cell. A. I have a solution of DNA that has a transmittance of 0.15 when it is placed in a cell with a 200 um pathlength. What is the concentration of DNA in this solution? (in units of g/ml) B. Assume that this DNA is a synthetic oligo containing 25 base pairs, and that the MW of a single base pair has a molecular weight of 330. What is the molar concentration of duplex DNA molecules in the solution? C. What is the Molar absorptivity of DNA in units of L cm (mol base pair) -1 -1
You have an extract of BL21(DE3) cells containing a soluble, 75 kDa recombinant protein that has a histidine tail at its C-terminus. This extract is still in raw form and you need to purify this protein without losing its solubility. Check the option below that represents the best purification methodology in the order of execution. * a) (1) isoelectric precipitation; (2) microfiltration; (3) affinity chromatography; (4) anion exchange chromatography b) (1) centrifugation; (2) microfiltration; (3) isoelectric precipitation; (4) affinity chromatography c) (1) centrifugation; (2) microfiltration; (3) affinity chromatography; (4) ultrafiltration d) (1) microfiltration; (2) jumping out; (3) centrifugation; (4) affinity chromatography
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Compare the melting temperature of a 1-kb segment of DNA containing20% A residues to that of a 1-kb segment containing 30% A residues under the same conditions.
ii only
2.21 The two allelic fragments of DNA shown belowa (d) After 30 cycles of O (b) Diagram the gel pattern you would expect to amplified by PCR, and the products digested with d St (a) What will the total sizes of the two PCR are JorX BA both PstI and Hind3. products be? from the doubly digested DNA.
SDS-PAGE and Agarose gel electrophoresis can both be used to separate proteins or protein- complexes based on their size. Separation of a multi-subunit protein complex by SDS-PAGE resulted in two bands with molecular weights of 86 KDa and 136 KDa, while separation of the same complex using Agarose gel electrophoresis resulted in two bands with molecular weights of 222 KDa and 444 KDa. Based on this information, which of the following statements is most likely to be correct? O The complex has a molecular weight of 222 KDa O The complex exists as a dimer of homo-dimers O The complex exists as a dimer of hetero-dimers SE O The complex exists as a trimer, but the individual protein subunits are covalently crosslinked to each other
Consider the equation below. What are its consequences in the context of an electrophoresis experiment? L In- 3πη K What property of DNA allows this equation to be valid? What must be done to proteins for this equation to be valid?

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