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Flashcards in Lecture 2 protein Furci Deck (51):
1

Slide 1 machine

ACCA - allows for specific protein purification despite time and difficulty. When protein needs to be de-bonded from organelle

2

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

3

Endotoxins

Can break down protein as we get to pure form.

4

Dimers, multimers, aggregates

Improper folding/bonding leads to this.

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Lysozymes present

Can degrade protein of interest.

6

General purification strategy

Characteristics: Solubility, Ionic charge, Polarity, Molecular Size, Binding Specificity

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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.

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Ionic Charge

Ion exchange chromatography, electrophoresis, isoelectric focusing

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Polarity

Adsorption chromatography, Paper chromatography, reverse phase chromatography, hydrophobic interaction chromatography

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Molecular size

Dialysis and ultrafiltration, gel electrophoresis, gel filtration/size exclusion chromatography

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Binding specificity

Affinity chromatography

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Salting in

Protien solubility increases with ionic strength, salt shields other protein charges (interactions). Basically allows for protein to be in functional state.

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Salting out

Solubility decreases as you increase ionic strength.

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Chromatography to purify

Size, Surface charge, Biorecognition (ligand specificity)

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Size chromo

Size exclusion chrom - gel filtration

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Surface charge chromo

Ion exchange chromo

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Biorecognition chromo

Affinity chormatography.

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Hydrophobic chromo

Exactly what is sounds like

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Chromatography resins/matrices

Sepharose, agarose, dextran. Different ligands, rigidity, bead and pore sizes can be used.

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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.

21

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.

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Ion exchange

Most easy process to use for purification. 1 step process. Has a protein matrix with albumin and immunoglobulin at pH 7.5.

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Resin for ion exchange

Cation exchanger (carboxymethylcellculose - charge), anion exchanger (diethylaminoethyl (DEAE) cellulose + charge).

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Albumin

pI 4.8 - charged

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Ig

pI of 8, + charged

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Protein mix at DEAE column at pH 7.5, with Ig and Albumin

Ig flows through, albumin to column.

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Removal from Ion exchange

Chemical breaks bond, then dialyze out.

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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.

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Purification of Immunoglobulin G IgG

Blood - plasma - antibodies - IgG. IgG interacts with rProtein A - use affinity chromo.

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IgG

Antibody - interacts with rProtein A through hydrophobic interactions on heavy and light chains.

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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.).

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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.

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Electrophoresis

Quick and easy way to separate by size/charge. SDS-PAGE usually used.

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SGS-PAGE *amend*

Slide 19

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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.

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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)

37

Protein hydrolysis

6M HCl, 110 degC, 24-48 hours

38

Determination of primary structure

Liquid chromatography, electron spray, ionization mass spec.

39

MALDI-TOF

Fastest method for primary protein structure. Iaser hits sample, ions accelerated and detected.

40

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)

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B. clostridium toxins.

Seven related toxins (serotypes A to G) produced by anaerobic bacillus Clostridium botulinum
Most potent toxins known (

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Food-borne

Toxin types A, B, E, (F). A is highest mortality, B is lowest mortality.

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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

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Infant BoTN

Intestinal colonization of organisms in infants younger than 1 year
Nearly all serotype A

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Inhalation BoTN

Laboratory accident

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Toxoid

Non-toxic preparation of bacterial toxin, Retains antigenic properties and elicits neutralizing responses against native toxin
Formalin (formaldehyde, glutaraldehyde) treatment

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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

48

New vaccine work

Expensive!

49

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)


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BOTOX

Botulinum toxin acquisition. Low concentration that tightens skin.

51

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