2) Protein Purification

what are the considerations of protein purification

source of protein

pH (avoid extreme pH values)

expression system (bacterial is simplest (consider gram+ or gram-)

tags (affinity tags)

pI of protein of fusion construct

cleavage of tag iwth protease

lysis conditions

second affinity chromatography step

how to get rid of fusion tag once it is cleaved

what are considerations with source of protein?

endogenous vs recombinant
endogenous = protein coming from native tissues/cells
recombinant = protein is over-expressed in expression system

tissue lysates vs expression systems
purifying from tissue lysates requires removing many other cellular proteins
recombinant expression systems may allow for simpler purification using engineered tags

what are types of expression systems? and details on them?

bacterial: (e coli) common for rapid expression and cost-effectiveness, but some proteins (eukaryotic), may not fold correctly or undergo proper post-translational modifications (PTMs) (look at gram+ or gram-)

yeast: (pichia pastoris), allows for some eukaryotic PTMs like glycosylation, but is still relatively fast and cost-effective. yeast glycosylation is eukaryotic but not necessarily human-like

insect cells: (baculovirus) offers a more complex environment for PTMs and protein folding, typically used for expressing proteins with more complex folding requirements

mammalian cells: (HEK293, CHO) ideal for proteins requiring human-like PTMs, but more expensive and slower than bacterial or yeast systems

what does gram + or gram - mean

what does this difference affect?

gram+ have thick wall made of sugar and protein (peptidoglycan), and no outer membrane (staph and strep)

gram- have a thin wall of peptidoglycan hidden under extra outer protective layer (e coli and salmonella)

affect how cells are lysed and what contaminants are released

what are the most common types of tags?

maltose binding

glutathione-s-transferase

6xHis

what is maltose binding protein (MBP)?

enhances solubility and is used in fusion with protein that tend to aggregate

binds to amylose/maltose for affinity purification

increases expression, used as an affinity tag

does not necessarily mean the target is correctly folded

binds amylose resin: free maltose competes and elutes MBP-tagged protein

what is glutathione-s-transferase (GST)?

solubility enhancing tag that binds glutathione (GSH) for affinity purification

dimeric, and can influence apparent oligomerization

GST: binds glutathione (GSH) for affinity purification. elution with reduced glutathione under mild conditions or with low pH

what is a 6xHis tag?

short polyhistidine tag that binds to nickel, cobalt, and zinc resins

highly versatile and commonly used due to its small size and ease of use

6xHis: binds to nickel, cobalt, or zinc chelate columns (eg. Ni-NTA) typically eluted competitively with imidazole; lowering pH is an alternative

what is pI?

the pH at which negative and positive charges on the protein are balanced

gives clues on which pHs the protein will prefer

if the protein is very positively charged, cells have dna and rna that are negatively charged and will bind together, not good

explain how to compare buffer pH and protein pI

if the pH is greater than pI, proteins net charge is negative, so you use an anion exchanger column which is positively charged t o attract negative protein

if pH is less than pI, protein’s net charge is positive, you use cation exchanger column which is negatively charged to attract positive protein

why is a protein with pI of 4 negatively charged at pH=7?

at pH = 4, protein’s positive and negative charges balance to net 0

when we increase pH to 7, the conc of H+ in solution decreases
this favours loss of protons from proteins ionizable groups (deprotonation)

the loss of H+ makes groups more negative, making the protein’s charge negative overall

some amino residues may remain positively charged (basic lysine and arginine) but the net charge is negative

what are considerations with lysis conditions?

buffer pH, salt, reducing agent, protease inhibitors

what do you need to consider for buffer pH in lysis conditions

pH can affect protein stability and solubility

should match optimal conditions for you protein

what do you need to consider for salt concentration in lysis conditions

affects protein solubility and interactions

some proteins need high salt to prevent aggregation

optimal salt conc depends on protein and purification method

what do you need to consider for reducing agents in lysis conditions

most protein like to be in a reducing environment, when you open them up, they are exposed to oxygen, so you might want to add reducing agents

cysteines are very sensitive to oxidation, need reducing agents, or will oxidize/form disulfides

what do you need to consider for protease inhibitors in lysis conditions

to prevent degradation of target proteins by endogenous proteases during lysis and purification process

how do you get rid of a fusion tag once its cleaved?

goal is purity, keep protein get rid of tag

MDP binds to amylose resin
GST binds to glutathione (GSH)
6xHIs binds to nickel, cobalt, and zinc

after tag is cleaved, it can still bind to the same affinity resin, then you pass protein mixture through the same or second column to remove the tag, leaving the target protein in the flow-through

what is second affinity chromatography step

after tag cleavage, second affinity chromatography step can be used to purify the target protein from the cleaved tag and the protease itself

using a resin that the cleaved tag will bind to, while the protein flows through

this works when the cleaved target is now untagged, while the cleaved tag and preferably the protease bind the affinity resin

His-tagged TEV makes this especially convienient

what is unique about TEV cleavage?

it’s cleavage sequence is ENLYFQ^G/S, so it leaves extra residues before the protein of interest

what are the most common separation methods

IMAC: immobilized metal affinity chromatography

IEC: ion-exchange chromatography (anion or cation)
cation chromatography catches something positively charged, so the resin must be negatively charged

SEC: size exclusion chromatography
often used no matter what as quality control. it’s the only technique that tells you the protein is properly folded

what are 3 protein requirements

metals: some proteins require metal ions for structural stability of enzymatic activity. some metalloproteins may requires the appropriate metal during purification to maintain occupancy, stability, or activity

co-factors: proteins that interact with cofactors (small molecules) during biological function may require them for proper folding or activity

post-translational modification: phosphorylation, glycosylation, or lipidation may be required for proper folding of function, especially in mammalian proteins

what is hydrophobic interaction chromatography

separates proteins according to exposed hydrophobic surface

relatively high salt concentration strengthens association between hydrophobic patches on the protein and the resin
decreasing salt conc weakens the interaction and elutes proteins in order of increasing hydrophobicity

less hydrophobic proteins emerge first in higher salt

uses mild aqueous conditions and preserves native structure

UV absorbance: presence of tryptophan and tyrosine

each one comes off with less salt than the last

lower salt = lower hydrophobicity interactions meaning they get eluted then

what is the impact of salt on protein purification

at low-modest ionic strength, salt ions shield charged groups on the surface of the protein which reduces the nonspecific electrostatic attraction between protein molecules, stabilizing and increasing solubility
“salting-in”

at sufficiently high salt conc, salt ions compete strongly for water, protein hydration decreases (less water on hydrophilic regions), protein-protein association and precipitation become more favourable,
“salting-out”

histone proteins on dna love salt

there is a window of moderate ionic strength where the protein solubility increases, but above and below this conc the solubility lowers (proteins interact more and stick together)

what is ammonium sulfate fractionation

used for salting out

highly soluble, inexpensive, stabilizes proteins

salt is added sequentially to generate crude protein fractions. after each salt increase, centrifugation separates proteins that precipitated within that interval into pellet, and proteins that remain soluble stay in supernatant

how much salt is needed is needed depends on starting saturation and temperature

explain details on case study 1: purifying 6xHis-calmodulin (cytosolic protein)

what should you look for in a case study example like this?

construct has N-terminal His tag and TEV cleavage site followed by calmodulin
fusion is small and acidic, pI=4.39

scan for cysteines (will need to add reducing agent)
scan for weird regions (lots of lysines 6-8, between 2 domains will be floppy, less stable, you would want to remove that to be more flexible)

theoretical pI because it depends on which amino acids are on the outside, can be off by as much as a whole unit, on exams can be within 0.5

what is step 1 on case study 1

e coli cell lysis with IMAC

add glycerol: very viscous, keeps proteins happy and doesn’t impede protein function

add mgcl: required for DNAse1
DNAse digests DNA out into smaller pieces, also helping protein function

EDTA free inhibitors, don’t want to accumulate
EDTA removes metal, but you want to keep the metal because nuclease activity requires Mg

what is step 2 in case study 1

IMAC: immobilized metal affinity chromatography

filtered supernatant from previous step loaded onto Nickel resin column
low conc of imidazole in wash step to get rid of any nonspecific binding

genetically encoded and translated is the tag, TEV recognizes the site of the tag and cuts it

why are imidazole, nucleases, lysozymes, and protease inhibitors used?

imidazole: used as competitive eluting agent to release His-tagged proteins from IMAC resins like nickel cobalt, reduce non-specific binding to IMAC

nucleases: break down contaminating host-cell DNA and RNA that interfere with processing and final quality of protein

lysozymes: break open bacterial cell walls so that target proteins trapped inside can be released in bacterial purification

protease inhibitors: prevent endogenous cellular proteases from breaking down and destroying target protein when cells are lysed

how is pH used during protein purification

controlling protein charge by taking into account pI

selective elution, changing pH during run alters protein’s charge, causing it to detach form chromatography column

in IMAC, adjusting pH helps weaken or strengthen interactions between tags and the resin (especially His-tag)

washing: prevent non-specific contaminants from sticking while washing

avoiding pI: at pI, protein carriers no net charge, minimizing solubility and often causes the protein to clump together and precipitate

why do we check soluble and insoluble fractions

determines whether target protein is dissolved in the cell liquid or trapped in clumps, advises how to proceed with extraction

if protein stays dissolved in supernatant/soluble fraction, you can move straight to standard purification like column chromatography

if protein forms solid clumps in pellet/insoluble fraction, it may mean the protein has folded incorrectly
if this happens you can add detergents, change the temperature, or use refolding step

details on IMAC

engineer a target gene to add affinity tag, usually His-tag to the protein

chromatography column is packed with solid support resin coated with chelating agent (NTA or IDA) that holds transition metal ions tightly (Nickel or Cobalt)

fluid containing his-tagged protein passes through column, histidine side chains form strong coordinate bonds with immobilized metal ions
unwanted proteins and contaminants pass through
buffer washes away any remaining non-specific or loosely attached impurities

imadizole is added to the column, mimics the structure of histidine and competes for the metal ion binding sites, releases the purified protein from the resin

TEV can be used to cleave his-tag off, using same process as original IMAC

what is ioin exchange chromatography

cation exchange means catching cations, resin is negative

anion exchange means catching anions, resin is positive

can gradually change ionic strength or the pH of the elution buffer, molecules are released from the resin at diff rates depending on strength of interactions, allowing for separation and elution

at binding step we don’t want salt, which will outcompete and bind to the column instead of the protein
to get low salt, you use osmosis, low conc of salt in solution outside, salt moves out then you are left with a lower conc of salt in the solution inside

explain these lanes

lane 1: cell pellet

lane 2: lysate (soluble part) at 19 kDa (this is the molecular weight of the protein)

lane 3: IMAC flowthrough, stuff that didn’t bind all throughout, contaminants

lane 4: IMAC elution, more of protein

explain lanes 5-7 here and extrapolate for 8-10

lane 5: add TEV digest, no purification has happened here, 27kDa is TEV, 19 kDa is CaM with His-tag, 17 kDa is CaM (purified)

lane 6: second IMAC/post-TEV IMAC flowthrough
flowthrough has untagged proteins of interest.

lane 7: elution, everything else which has a histag and we don’t care about that

lane 8: IEC, pI is 4.4, we use buffer of pH=7 and anion exchange.

lane 9: elution at kDa 17

lane 10: size exclusion, assume protein will come through and draw a line at the intended size

why would you use buffer of pH=7 on a pI of 4

use a pH that’s at least 0.5 away from the pI

keep it close to physiological pH to not denature the protein

basically just use pH=7 no matter what unless pI is between 6.5 and 7.5

explain steps of case 2

this kinase has cystines, it needs reducing agent

1) IMAC with TCEP (breaks sulfide bonds without removing metals from column) IMAC will not work if nickel is reduced

2) TEV cleavage (TEV is also his-tagged), the purpose is to make protein more soluble and boost expression

3) post-tev amylose resin (gets rid of tag but not TEV), amylose binds to kinase, elute with maltose

4) IMAC and collect FT (gets rid of tag and tev, uncleaved fusion should bind)

5) IEC, protein kinase, so use buffer at pH=7, protein is positively charged at that pH
need to do dialysis first, reduce salt before IEC

what are the simplified steps for protein purification

1) IMAC with TCEP (with reducing agent if C present)

2) TEV cleavage (make protein more soluble and boost expression)

3) Post-TEV amylose resin, (captures MBP tag no longer connected to protein), eluted with maltose,

4) IMAC, (gets rid of tag and TEV, uncleaved protein should bind and you collect FT)

5) IEC, do dialysis to remove salt interference, know buffer pH (close to 7 regardless, and cation/anion)

6) SEC, tells you the protein is properly folded, quality control

explain this

as salt increases, conductivity increases

kinase is in Peak 1 and Peak 2

third peak is TEV, which lingers around even after all the steps

explain each lane, what are the white boxes?

boxes (in order): kinase, fusion (uncut), MBP, TEV, kinase

1: IMAC elution
2: TEV cleavage

3: PostTEV amylose resin FT
4: PostTEV amylose resin elution (get uncut fusion and MBP tag)

5: IMAC FT, collect protein)
6: IMAC elution (TEV still there plus things bound to nickel)

7: IEC FT, lose a tiny bit a protein in FT
8: IEC Elution peak 1
9: IEC Elution peak 2

why would the protein kinase have 2 different peaks in IEC elution

protein kinase is regulated by phosphorylation, some molecules may be more or less phosphorylated

more phosphorylation means the protein is slightly more negatively charged and would elute at a different salt conc (higher for anion, lower for cation)

what is SEC?

size exclusion chromatography

separates proteins according to hydrodynamic size, not molecular mass alone

protein shape matters: elongated protein can elute earlier than compact globular protein of the same mass

SEC resins consist of porous matrix of beads that lack reactivity and adsorptive properties
molecules larger than pore size are unable to diffuse INTO the beads and elute first
most cases these are aggregated proteins (we don’t want), will create aggregate peak

if molecule is smaller than the smallest pores in the resin, they elute last

explain details on SEC

good result: normal distribution of protein at one particular time, but beware, co-eluting species can remain hidden beneath one peak, so one sharp peak does not demonstrate purity by itself

bad result: things aren’t separating well, there’s multiple peaks and wobbly lines throughout

what’s another way to purify CaM?

use the function of the protein as a purification step

bind to calcium first, which makes it sticky and will bind to column

to get off column, apply EDTA, which chelates Ca2+, reversing hydrophobic state of CaM, promoting elution of CaM