Flashcards in Lecture 2 protein Furci Deck (51):
Slide 1 machine
ACCA - allows for specific protein purification despite time and difficulty. When protein needs to be de-bonded from organelle
Biochemical investigations usually require pure components
Typical cell contains thousands of different substances
Many biomolecues have similar physical and chemical properties
Biomolecueles may be unstable and/or present in vanishingly small quantities
Purification of biomolecules is a formidable task
Would be considered unreasonable difficult by most synthetic chemist
Can break down protein as we get to pure form.
Dimers, multimers, aggregates
Improper folding/bonding leads to this.
Can degrade protein of interest.
General purification strategy
Characteristics: Solubility, Ionic charge, Polarity, Molecular Size, Binding Specificity
Salting in/out - adjust soln. to just below point of solubility for protein. Change ionic strength (add salt), change polarity (add organic solvent), change pH + temp can do this. Problem: precipiate other proteins. Separate soluble and insoluble material by centrifugation/filtration. Usually step 1.
Ion exchange chromatography, electrophoresis, isoelectric focusing
Adsorption chromatography, Paper chromatography, reverse phase chromatography, hydrophobic interaction chromatography
Dialysis and ultrafiltration, gel electrophoresis, gel filtration/size exclusion chromatography
Protien solubility increases with ionic strength, salt shields other protein charges (interactions). Basically allows for protein to be in functional state.
Solubility decreases as you increase ionic strength.
Chromatography to purify
Size, Surface charge, Biorecognition (ligand specificity)
Size exclusion chrom - gel filtration
Surface charge chromo
Ion exchange chromo
Exactly what is sounds like
Sepharose, agarose, dextran. Different ligands, rigidity, bead and pore sizes can be used.
Dailysis and ultrafiltration
Pore size for small molecules - stir bar pulls out smaller molecules, leaving larger ones inside. Intermediate step helping to get to purified product.
Exactly what it sounds like. Protein goes through gel polymer beads, larger proteins fall through quickly, smaller protein gets stuck up in different pores. EX. Phenylalanine and other UV vis proteins allow for UV absorption, so you can see where your proteins are going. Salt will be removed last.
Most easy process to use for purification. 1 step process. Has a protein matrix with albumin and immunoglobulin at pH 7.5.
Resin for ion exchange
Cation exchanger (carboxymethylcellculose - charge), anion exchanger (diethylaminoethyl (DEAE) cellulose + charge).
pI 4.8 - charged
pI of 8, + charged
Protein mix at DEAE column at pH 7.5, with Ig and Albumin
Ig flows through, albumin to column.
Removal from Ion exchange
Chemical breaks bond, then dialyze out.
1 step, best selectivity, nearly pure in 1 step. Specific interaction of substrate with ligand. Be sure to have enough resin to do this, as protein will slowly trickle down.
Purification of Immunoglobulin G IgG
Blood - plasma - antibodies - IgG. IgG interacts with rProtein A - use affinity chromo.
Antibody - interacts with rProtein A through hydrophobic interactions on heavy and light chains.
Bump is unwanted protein. Change in elution buffer stimulates release of desired product. Now we have protein in elution buffer, we want it in storage buffer (adjust , add stabilizers, pH, liquid or dry etc.).
Example of buffer exchange
Know that once protein comes off, you need to get it into an ideal state, and that there are numerous (3 shown) ways to do this.
Quick and easy way to separate by size/charge. SDS-PAGE usually used.
Separates protein on the basis of pI. 1 dimension is SDS-PAGE, other is IEF from pH 4 to 7. Separates by size and charge.
Order of purification for sequencing
Extraction - salt out/dialysis + concentration - ion exchange or gel filtration - SDS PAGE - Proteolytic degradation (use enzymes to cut at sites bromyline) - peptide purification and sequencing (chromatogram used)
6M HCl, 110 degC, 24-48 hours
Determination of primary structure
Liquid chromatography, electron spray, ionization mass spec.
Fastest method for primary protein structure. Iaser hits sample, ions accelerated and detected.
Anaerobic, produces endospores, 30 species responsible for human infection, >50 species found in environment. Proteins proteinaceous toxins that cause disease symptoms. Clostridium bltulinum (food poisoning)
B. clostridium toxins.
Seven related toxins (serotypes A to G) produced by anaerobic bacillus Clostridium botulinum
Most potent toxins known (
Toxin types A, B, E, (F). A is highest mortality, B is lowest mortality.
Systemic spread of toxin produced by organisms inhabiting wounds, trauma, surgery, subcutaneous heroin injection, and sinusitis from intranasal cocaine abuse
Types A and B
Intestinal colonization of organisms in infants younger than 1 year
Nearly all serotype A
Non-toxic preparation of bacterial toxin, Retains antigenic properties and elicits neutralizing responses against native toxin
Formalin (formaldehyde, glutaraldehyde) treatment
Pentavalent Botulinum Toxoid (PBT) Vaccine
Combination of formalin-inactivated type A,B,C, D, and E botulinum toxins; (separate vaccine: divalent F and G)
Aluminum phosphate-adsorbed with formaldehyde and thimerosal used as preservatives
New vaccine work
Neutralize circulating BoTN toxin. Bivalent botulinum antitoxin (serotypes A and B) is the only FDA-approved antitoxin available. Trivalent equine botulinum antitoxin (serotypes A, B, and E) is no longer available . Heptavalent antitoxins against Types A through G available as investigational (IND) products from the USAMRIID. BabyBIG approved by the FDA for treatment of infants with botulism from toxin serotypes A and B
Human botulism immune globulin derived from pooled plasma of adults immunized with PBT (A through E)
Botulinum toxin acquisition. Low concentration that tightens skin.