protein purification Flashcards
(34 cards)
What is one of the great promises of the biotechnology industry?
The ability to produce biopharmaceuticals to treat human disease
Who pioneered the development of recombinant DNA technology?
Genentech
What was insulin traditionally extracted from before recombinant DNA technology?
Pancreas glands of swine and cattle
How many pounds of animal pancreas glands were needed to produce one pound of insulin?
Approximately 8,000 pounds
What significant product did Genentech produce using recombinant DNA technology?
The world’s first commercial genetically engineered product: human insulin
What challenges are faced when producing novel proteins in bacteria?
Active proteins are often comprised of multiple chains of amino acids with complex folding and strand interactions
What are some considerations in developing a recombinant protein?
- Cell type
- Plasmid construction
- Purification strategy
What does FPLC stand for?
Fast Protein Liquid Chromatography
What are the columns used in FPLC composed of?
Small particles or gel beads known as stationary phase
What is the purpose of the chromatographic bed in FPLC?
To separate the sample mixture based on different components adhering to or diffusing through the gel
What types of separation can FPLC columns perform?
- Size
- Charge distribution (ion exchange)
- Hydrophobicity
- Reverse-phase
- Biorecognition (affinity chromatography)
What is a notable feature of FPLC columns compared to HPLC columns?
FPLC columns can only be used up to a maximum pressure of 3-4 MPa (435-580 psi)
Can FPLC columns be used in an HPLC machine?
Yes, if the pressure of HPLC can be limited
Fill in the blank: Genentech engineered bacterial cells to produce _______.
human insulin
True or False: FPLC and HPLC use the same type of columns without any limitations.
False
What was the first recombinant protein used in treatment and in what year?
Human Insulin (1982)
What collaboration in July 2018 produced a PD-L1 antibody for immuno-oncology research?
Abcam Merck collaboration
What are the three main phases in protein purification workflow?
- Capture Phase
- Intermediate Purification Phase
- Polishing Phase
What is the first step in protein extraction, fractionation, and enrichment?
Tissue or cell lysis
List two mechanical methods used in tissue or cell lysis.
- Sonication
- Grinding
What can be added to lysis reagents to prevent proteolytic activity?
Protease and phosphatase inhibitors
At what temperature range are most proteins stable?
0-4 ºC
What does the Hofmeister series classify?
Ions in order of their ability to salt out or salt in proteins
Fill in the blank: Proteins are soluble in aqueous media because they have _______ amino acid side-chains.
hydrophilic
