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Questions and Answers
Which of the following cell types would be MOST appropriate for expressing a complex human protein requiring extensive post-translational modifications?
Which of the following cell types would be MOST appropriate for expressing a complex human protein requiring extensive post-translational modifications?
- Bacterial cells, due to their rapid growth rate.
- Insect or mammalian cells, capable of complex protein processing. (correct)
- Yeast cells, offering a balance between culture ease and processing.
- All cell types are equally equipped for complex protein modification.
A researcher aims to purify a protein from a plant tissue sample. Which disruption method is MOST suitable?
A researcher aims to purify a protein from a plant tissue sample. Which disruption method is MOST suitable?
- French press, due to its high-pressure cell lysis.
- Enzymatic digestion, effective on cell cultures without cell walls.
- Sonication, due to its effectiveness on rigid cell walls.
- Homogenization, due to its suitability for tissues. (correct)
Following cell disruption and centrifugation, where would the target protein be found if it is soluble?
Following cell disruption and centrifugation, where would the target protein be found if it is soluble?
- Attached to the disruption equipment.
- In the supernatant, the liquid after centrifugation. (correct)
- Protein is lost and the experiment must be repeated.
- In the pellet, as the solid precipitate collected.
Which of the following statements accurately compares mechanical and non-mechanical cell disruption methods?
Which of the following statements accurately compares mechanical and non-mechanical cell disruption methods?
If a protein is produced recombinantly in E. coli, which method would be LEAST suitable for the initial cell lysis step?
If a protein is produced recombinantly in E. coli, which method would be LEAST suitable for the initial cell lysis step?
A researcher is working with a protein that is prone to denaturation under high pressure. Which cell lysis method should they avoid?
A researcher is working with a protein that is prone to denaturation under high pressure. Which cell lysis method should they avoid?
A researcher is purifying a protein expressed in yeast cells. After cell disruption, they observe a very viscous lysate. Which issue is MOST likely causing this?
A researcher is purifying a protein expressed in yeast cells. After cell disruption, they observe a very viscous lysate. Which issue is MOST likely causing this?
A protein purification protocol includes bacterial cell lysis, centrifugation, and affinity chromatography. After affinity chromatography, SDS-PAGE reveals several unexpected bands along with the target protein. What step should be optimized FIRST?
A protein purification protocol includes bacterial cell lysis, centrifugation, and affinity chromatography. After affinity chromatography, SDS-PAGE reveals several unexpected bands along with the target protein. What step should be optimized FIRST?
Flashcards
Protein Purification
Protein Purification
The process of isolating proteins from a mixture for study.
Four-step Procedure
Four-step Procedure
Protein purification generally follows these steps: Source, Disruption, Clarification, Refinement.
Sources of Protein
Sources of Protein
Sources can be natural (plants, animals) or recombinant (bacteria, yeast).
Mechanical Disruption
Mechanical Disruption
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Homogenization
Homogenization
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Sonication
Sonication
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French Press
French Press
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Enzymatic Digestion
Enzymatic Digestion
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Study Notes
Protein Purification
- Protein purification is crucial for biological, physiological, and pharmaceutical research.
- Protein purification isolates specific proteins from a mixture.
- It aids in protein function studies, drug development, and understanding disease mechanisms.
- Protein purification commonly involves four steps: (1) Source selection, (2) Cell disruption, (3) Clarification, and (4) Refinement.
- Protein sources can be natural (e.g., plants, animals) or recombinant (produced in cells via genetic engineering).
- Recombinant proteins are produced in various cell types, including bacteria, yeast, insect cells, and mammalian cells.
- Bacterial cells are easily cultured but lack complex eukaryotic protein processing.
- Yeast cells offer a balance between ease of culture and protein processing capabilities.
- Insect and mammalian cells are preferred for complex proteins, though they are more challenging and expensive to cultivate.
- Cell disruption is necessary to extract proteins from cells or tissues, either natural or recombinant.
- Disruption methods include mechanical (homogenization, sonication, French press) and non-mechanical (enzymatic digestion) techniques.
- Homogenization forces the sample through a narrow space, similar to a blender.
- Sonication generates ultrasonic waves to shear cell walls and DNA.
- The French press uses high pressure to rupture cells by bursting them open.
- Enzymatic digestion utilizes enzymes like lysozyme to break down cell walls, followed by detergent treatment of remaining components.
- Clarification, often by centrifugation, separates the protein-containing liquid (supernatant) from the solid cellular debris (pellet).
Homogenization
- Homogenization is widely used for plant and animal tissues.
- The process operates like a blender, guiding the sample against a spinning metal rod, breaking tissue against an impact ring.
Sonication
- Sonication is beneficial for cellular protein sources.
- A metal probe vibrates rapidly, creating ultrasonic sound waves for cell wall and DNA shearing.
French Press
- The sample is pressurized in a metal chamber, with pressure release rupturing cells.
Enzymatic Digestion
- Enzymatic digestion, suitable for cells with walls (bacteria, yeast, plants), uses enzymes like lysozyme to break down cell walls.
- Detergents solubilize remaining cell components.
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Description
Protein purification is essential for biology, physiology, and drug development, involving isolation of proteins from mixtures to study their function, develop drugs, and understand disease mechanisms. The procedure includes sourcing, disruption, clarification, and refinement, using natural or recombinant sources like bacteria, yeast, insect, and mammalian cells.
