Lecture 2 protein Furci Flashcards
Slide 1 machine
ACCA - allows for specific protein purification despite time and difficulty. When protein needs to be de-bonded from organelle
Protein extraction
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
Endotoxins
Can break down protein as we get to pure form.
Dimers, multimers, aggregates
Improper folding/bonding leads to this.
Lysozymes present
Can degrade protein of interest.
General purification strategy
Characteristics: Solubility, Ionic charge, Polarity, Molecular Size, Binding Specificity
Solubility (purification)
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.
Ionic Charge
Ion exchange chromatography, electrophoresis, isoelectric focusing
Polarity
Adsorption chromatography, Paper chromatography, reverse phase chromatography, hydrophobic interaction chromatography
Molecular size
Dialysis and ultrafiltration, gel electrophoresis, gel filtration/size exclusion chromatography
Binding specificity
Affinity chromatography
Salting in
Protien solubility increases with ionic strength, salt shields other protein charges (interactions). Basically allows for protein to be in functional state.
Salting out
Solubility decreases as you increase ionic strength.
Chromatography to purify
Size, Surface charge, Biorecognition (ligand specificity)
Size chromo
Size exclusion chrom - gel filtration
Surface charge chromo
Ion exchange chromo
Biorecognition chromo
Affinity chormatography.
Hydrophobic chromo
Exactly what is sounds like
Chromatography resins/matrices
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.
Size exclusion
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.
Ion exchange
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).
Albumin
pI 4.8 - charged
Ig
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.
Affinity chromo
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.
IgG
Antibody - interacts with rProtein A through hydrophobic interactions on heavy and light chains.
Slide 15
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.
Electrophoresis
Quick and easy way to separate by size/charge. SDS-PAGE usually used.
SGS-PAGE amend
Slide 19
Isoelectric Focusing
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)
Protein hydrolysis
6M HCl, 110 degC, 24-48 hours
Determination of primary structure
Liquid chromatography, electron spray, ionization mass spec.
MALDI-TOF
Fastest method for primary protein structure. Iaser hits sample, ions accelerated and detected.
Clostridium sp.
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 (
Food-borne
Toxin types A, B, E, (F). A is highest mortality, B is lowest mortality.
Wound
Systemic spread of toxin produced by organisms inhabiting wounds, trauma, surgery, subcutaneous heroin injection, and sinusitis from intranasal cocaine abuse
Types A and B
Infant BoTN
Intestinal colonization of organisms in infants younger than 1 year
Nearly all serotype A
Inhalation BoTN
Laboratory accident
Toxoid
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
Expensive!
Antitoxin
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)
BOTOX
Botulinum toxin acquisition. Low concentration that tightens skin.
Botulinum toxin purification
All procedures carried out in sealed containers in Class II/III Biological Safety Cabinets
Solubilize toxin complex from “mud” and precipitate
Acid wash steps to further purify and recover
Purify toxin complex by chromatography and precipitate/crystallize
Laboratory lyophilization produced poor results, thus pilot plant drying was not attempted
Liquid slurry used in testing
Storage: Low pH at or below 15°C
