Protein Purification/Bioprocessing

Question 1

protein engineering or enzyme engineering

Answer 1

creating entirely new protein molecules with desired properties

Question 2

bioprocessing

Answer 2

use of living cells to manufacture a product

Question 3

bioprocess in protein production**

Answer 3

2 phases in protein production (or any biological product):
1. upstream process: includes construction of expression vector, strain engineering, and culture optimization
2. downstream process: separation of the protein from cells and from other proteins (protein purification: liquid chromotography)

Question 4

upstream process vs. downstream process**

Answer 4

1. upstream process: isolate gene of interest, cut gene from chromosome, attach/ligate to a plasmid to create recombinant plasmid –> insert new plasmid into bacteria –> plasmid multiplication –> cell growth –> now you have small culture –> cells grown in bioreactor –> will scale up to eventually reach industrial scale
2. downstream process: starts after scale up step in upstream processing –> separate out product of interest from everything else in medium/host cell (entire cells harvested if protein is intracellular) –> centrifugation/separation step –> lyse it open and isolate all proteins from the interior of the cell –> then purify those; purify your protein of interest –> after purification needs to be tested out

Question 5

upstream process –> how do you optimize cell culture growth? how do you scale up?

Question 6

bioprocess in protein production**

Answer 6

2 phases in protein production (or any biological product):
1. upstream process: includes construction of expression vector, strain engineering, and culture optimization
2. downstream process: separation of the protein from cells and from other proteins (protein purification: liquid chromatography)

Question 7

chromatography

Answer 7

difference in protein chemical properties allows separation of target protein from other proteins
1. size exclusion chromatography: uses gel beads with pores (of different sizes) –> larger proteins bust through beads
2. ion exchange chromatography: relies on charge of protein –> resin is charged (positive or negative) –> oppositely charged proteins will stick to resin beads
3. affinity chromatography: proteins bind specifically and reversibly to uniquely shaped compounds called ligands (antibody-antigen interaction)
4. hydrophobic interaction chromatography: sorts proteins based on repulsion of water

(5. high-performance liquid chromatography: uses high pressure to force the extract through a column a shorter time
6. mass spectrometry: highly sensitive method used to detect size and identity of most protein fragments)

Question 8

processes that can analyze presence of protein during purification

Answer 8

1. SDS-PAGE
2. Western blot
3. ELISA

Question 9

post-purification analysis methods

Answer 9

1. liquid chromatography followed by mass spec (LC-MS): determines primary structure/amino acid sequence
2. nuclear magnetic resonance (NMR): determines tertiary and quaternary structures in solution phase
3. X-ray crystallography: determines tertiary and quaternary structures of crystals