MALDI-TOF = Matrix-Assisted LaserDesorption/Ionization – Time of Flight

MALDI-TOF Mass Spectrometry (MS)

MILK Protein purification

first step, so many times, on the way of elucidating protein structure and function: heterologous (inducible) protein expression and protein purification

heterologous: ‘derived from a different organism’, e.g. an eukaryotic protein expressed in Escherichia coli (E. coli, Gram-negative bacterium)

we put the DNA sequence of an ‘alien’ protein into a host that is able to express that protein as if it would be of its own (see recombinant DNA technology related details in molecular biology lectures later)

generally we talk about ‘over-expression’ because we try to use such a ‘vector’ that drives expression of the alien protein with a relatively high yield

vector = (generally) a plasmid specifically used for protein expression in the target

plasmid = a circular, double stranded DNA molecule that is separate from chromosomal DNA (of the host, e.g. bacteria) and can replicate indepen-dently (transferable genetic elements that can be found naturally in bacteria and in some eukaryotes; the size of a plasmid may be of 1 up to 1,000 kilobase pairs (kbp) and their number may be even thousands per cell)

in E. coli more complex proteins or proteins with “eukaryotic” post-trans-lational modifications cannot be expressed correctly

proteins can be targeted to the cytosol or the periplasm (leader sequence) and can be found in the form of inclusion bodies in either of the places (foradvantages/disadvantages of cyosolic/periplasmic expressions and refolding strategies, please, see previous lectures)

if correct post-translational modification of the protein is a necessity thenit is possible to use other hosts like insect cells (Baculovirus), yeast cells(Pichia (e.g. pastoris)), mammalian cells, and cell-free (in vitro) expression

proteins can sometimes also be efficiently purified from natural sources, like heart, liver, brain, etc. of various animals (including humans, but mostly ‘post-mortem’) or plants

proteins can be separated from one another on the basis of their solubility, size, charge and binding ability

Adage of Biochemistry: “Never waste pure thoughts on an impure protein”

the purified protein may even be only 1% of the original mass of cells, plantor animal sample

first things first: an assay is needed to prove the protein is present (gene-rally an activity assay of an enzyme)

A340 nm

we should also estimate how much protein of interest we have in the sample to purify

with activity and protein amount in hand we calculate specific activity (activity per unit mass) that is going to be increasing and has to be monitored during the purification process

the overall goal of protein purification is to maximize specific activity

first step of purification (from natural sources) is to fractionate cells intocomponents and determine which component is enriched in the protein of interest

cells can be fractionated by centrifugation after braking the cell membrane (sonication, French press, lysozyme, etc.): pellet (solid) and supernatant (liquor); repetitive centrifugation at increasing speeds (expressed in g-force or rpm) can yield further cellular fractions: differential centrifu-gation

separate fractions are tested for the protein of interest (activity, SDS-PAGE, monoclonal antibody in Western blot technique) and then the fractionwith the highest abundance of the protein in question will be purified further

protein purification generally consists of multiple consecutive steps of various kinds to result the purified protein in the end; each step may take advantage of one segment of protein properties, like charge, size, binding properties or solubility

at each step the protein of interest is assayed and the specific activity is determined

a variety of purification methods are available; the perfect combination ofpurification steps for a specific protein depends on the molecular proper-ties and the source where we wish to purify the protein from

Salting out of proteins

used for fractionating proteins based on their different solubilities in the actual solvent (buffer); precipitated proteins may be filtered out or centri-fuged and dissolved back in appropriate buffer (followed generally by dialysis) whilst supernatant can directly be used (generally also after dialysis) if the protein of interest is in this fraction (or poured away if not)

generally done with ammonium sulfate

also used for concentrating dilute proteins (but better nowadays to use pressure cells or ultra-filtration centrifugal devices having semi-perme-able membranes with appropriate molecular weigh cut-off (MWCO) built in to retain or even separate proteins

(see also lecture #3 for further details)

ultrafiltration devices:

Dialysis

it can separate proteins from denaturants (during refolding), salts (after salting out) or other contaminants including smaller proteins (have to choosea dialysis bag with appropriate MWCO)

it can be used for buffer-exchange between or after separation steps

used generally in consecutive dialysis steps with 1:50 to 1:1000 dilution ratios (~4 h each round with mixing the outer solution for better efficiency)

dialysis cassettes in modern laboratories for micro-samples (high recovery of samples)

Gel filtration (size-exclusion) chromatography

it is used to:separate proteins of dissimilar sizes

buffer exchange of protein solutions

determining multimerization state (oligomerization order)via running first standard proteins of known MW

the matrix is made of polysaccharide chains (dextran (Sephadex), agarose) or polyacrylamide containing chemical cross-links that forms globular beadsof ~100 m size in where inside the cross-links present a molecular mesh; different densities of the cross-links among polymer chains result in varyingsizes of holes in the matrix and different separation profiles

these same polymer chains are the scaffolds of anion- and cation-exchange chromatography column matrices as well where positive or negative func-tional groups are attached chemically to the matrix

Ion-exchange chromatography

the basis of separation is the net charge of the proteins in the mixture

binding to the matrix will depend on the pI of the protein and the pH of the running buffer; proteins with negative net charges will bind to positi-vely charged columns (anion-exchange chromatography) while positively charged proteins will bind to columns with negative functional groups on them (cation-exchange chromatography)

in a mixture of proteins with varying pIs at a set buffer pH, there will beproteins that will not bind to the column at all (e.g. positive protein to positive column); this is a pre-separation right away at the beginning of the experiment

proteins of opposite charges to the one of the column will bind but with different affinities depending on their charge densities; fractionation during elution is possible by changing the pH or the salt concentration (latter is more common applying gradient elution, salt ions will compete with protein-matrix Coulomb interations)

cation-exchanger column: carboxymethylcellulose (CM-cellulose)anion-exchanger column: diethylaminoethylcellulose (DEAE-cellulose)

Affinity chromatography

this technique takes advantage of the high affinity of proteins to variousagents (small molecules [substrates, ligands], other proteins, etc.)

for example, plant protein concanavalin A can be purified on a column thathas glucose attached to it covalently; most other proteins in a plant extractdo not bind glucose

elution of concanavalin A can be carried out by adding concentrated glucoseto the column that will compete with the glucose on the column for the protein and releases the protein from column

main steps of purification: 1. administration of protein mixture to thecolumn, 2. extensive washing with indifferent buffer to get rid of non-bonding and non-specifically bound proteins, 3. elution of protein of interest by excess interaction partner freely dissolved in the elution buffer

if a netural interaction partner is not specific enough for the particular protein to selectively isolate it through affinity chromatography then the protein can be expressed e.g. in E. coli with an N- or C-terminal tag (or the combination of multiple tags, e.g. His-tag, maltose-binding protein (MBP) tag, glutathion-S-transferase (GST) tag, Strep tag) designed for such purposes

Chromatography is done in various set-ups

column chromatography (ambient pressure and gravitational force): this can be semi-automated by delivering solvents to column by peristaltic pumps and collecting fractions by a fraction collector

FPLC (Fast Protein Liquid Chromatography): medium pressure system using synchronized pumps to deliver solvents; separates especially proteins in liquid state (higher pressure is needed due to smaller particle sizes of chromatographic matrix which delivers the higher efficacy in separation), also uses fraction collector, UV/VIS lamp for photometric detection, fully automated and computer operated

HPLC (High Performance or High Pressure Liquid Chromatography): high pressure system (~200 bar, but up to even 1000 bar: Ultra High PressureLC), protein structure generally does not tolerate such high pressures, mostly used for separating smaller molecules (organic molecules, meta-bolites, but with very high resolution [small particle sizes in matrix, 1-10 m, that is why the high pressure to be able to push the solvent through with reasonable elution times for molecules]) that necessitates the use of organic solvents (often solvent mixtures and their gradients) and apolar matrices (C18-columns), fully automated, computer operated

HPLC I fractionation of human urine Sephadex G-25 postsalt peak.

Cain C D et al. PNAS 2007;104:17873-17878

©2007 by National Academy of Sciences

Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE)

a technique useful for separating (mostly) analytical amounts of proteins for determining their MWs and the composition of the protein mixture

protein chains are denatured and separated (S-S bonds are reduced by DTT or -SH-EtOH) and bind the detergent dodecyl (lauryl) sulfate (amphipathic molecule) statistically at every second aa in a repetitive fashion showing the negative charges of the sulfates outwards of protein;the net charge is roughly proportional to the mass of the protein

this negatively charged protein mixture will all move towards the positiveelectrode upon appropriate potential difference in the electrophoresis unit

a main difference from gel filtration is that here ALL molecules are forcedthrough the same matrix, hence the bigger molecules will travel slower

after separation, proteins can be stained with Coomassie Blue (0.1 g sensi-tivity) or silver stain (0.02 g sensitivity)

MWs different in only 2% or in general of 10 aa can already be separated

we can use different density of cross-links here too (different pore sizes)that will affect the resolution power of the gel in various ranges of MW (even gradient gels can be poured); the upper part of the gel is poured separately and is called the stacking gel to form a thin starting zone of proteins before they enter the separating gel (because of the height of the sample loaded in the wells)

the log MW is inversely and linearly proportional to the electrophoretic mobility of proteins (except for some heavily glycosylated proteins) that can be used to determine MW; in most SDS-PAGE experiments a MW standard mixture of proteins is also loaded in one of the wells for refe-rencing MW

the protein pattern in the gel can be transferred to a membrane and used for immuno-detection using a monoclonal antibody raised against the protein(Western blot technique)

SDS-PAGE and Western blot pictures can be quantitated by densitometry

Isoelectric focusing

at the pI electrophoretic mobility is zero since there is no net charge of the protein

no SDS in gel but there is a pH gradient and proteins move during electro-phoresis until they reach a pH in gel that is close to their pI, then they stop moving

proteins differ in only 0.01 of pI can already be separated

2D gel electrophoresis

SDS-PAGE and isoelectric focusing (IF) can be combined to do a very high-resolution separation (combined further with tandem mass spectrometry is a state-of-the-art way of analyzing complex mixtures of proteins, e.g. healthy versus pathological tissue samples, basic technique of proteomics)

1. IF2. SDS-PAGE

> 1000 proteins from E. coli are separated