Molecules, genes and cells - PROTEIN PURIFICATION AND CHARACTERISATION

How to purify a protein from cells

1) Grow cells & obtain pellet.

2) Lyse cells to release proteins = homogenation.

3) Centrifuge to remove cell debris.

4) Differential Fractionation Methods

Differential Fractionation Methods

Reduce solubility of protein by changing the properties of the solution.

DFM types

Salt, Heat, Isoelectric

Salt fractionation

Ionic strength

Addition of ammonium sulfate. Competes with H20 precipitating protein.

Heat fractionation

Temperature

Protein of interest must be heat resistant. Increase temp. Heat labile will come out of solution.

Isoelectric precipitation

Changing of pH to pI. Precipitates protein.

Isoelectric focusing

Gel electrophoresis with pH gradient. Protein migrates to pH = pI.

pH < pI the protein is positively charged

pI

The isoelectronic point or isoionic point is the pH at which the overall surface charge of a protein is 0.

Types of column chromatography

Ion-exchange, affinity and size-exclusion.

Ion-exchange column

Positively charged beads.

Bind negative protein - equilibrium on/off action.

Positively charged elute faster. To elute negative, increase salt conc or pH.

Size-exclusion column

Porous beads.

Larger proteins elute first.

Affinity column

Most efficient.

Beads have covalently attached substrate so enzyme binds - equilibrium on/off action. Then elute protein by adding high concentration of competing substrate.

Ni 2+ beads

HexaHis tag bind.

How to know what is in each fraction eluted from column chromatography?

1) Spectrophometer

2) Gas chromatogram

3) Gel electrophoresis

OF EACH FRACTION

What happens to the specific activity and total activity during protein purification?

Throughout purification protein including POI is lost therefore total decreases but specific increases.

How to purify a protein based on SIZE & SHAPE

1) Ultracentrifugation

2) Gel electrophoresis

3) Size-exclusion chromatography

based on CHARGE

Isoelectric focusing

based on SOLUBILITY

Salt fractionation

based on ABILITY TO BIND TO OTHER MOLECULES

Affinity chromatography

SDS-Polyacrylamide Gel Electrophoresis

-SDS PAGE separates proteins on basis of mass, not charge

-SDS interrupts non-covalent interactions, binding to proteins and creates large negative net charges, neutralizing the protein's original net charge so only variable affecting velocity is frictional coefficient, which is dependent on mass

-gel stained after to visualize protein bands

SDS-PAGE

1 band on gel = 1 linear polypeptide

Native Polyacrylamide gel electrophoresis (PAGE)

PROTEIN NOT DENATURED, retains 3 & 4 structures.

Coomassive brill blue G-250 imparts negative charge without unfolding.

Two-dimensional gel electrophoresis

Separates proteins in two steps, first by isoelectric points and then by molecular weights.

Mass Spectrometry

m/z = mass charge ratio

ionised sample, vacuum

protein denatured and not recovered

Analytical ultracentrifugation

The ease at which something sediments.

Soluble proteins stay in solution & only sediment when subjected to large acceleration - sedimentation coefficient.

V ACCURATE PROTEIN MASS

Light scattering

I 0 --> sample --> I

the degree of scatter and intensity of light

I scattered = protein conc x molar MASS

overall effect from constructive and deconstructive interference for intensity

Spectroscopic techniques

Wavelength matters. EM spectrum. Gamma = smallest wavelength and highest frequency

Which spectroscopic techniques use wavelengths from UV?

Fluorescence, Absorption, Circular dichroism

Which spectroscopic techniques end up with a protein structure?

NMR ( radio )

X-ray crystallography

Absorption Spectrometry

E = hv

Excites electrons. Level of Abs changes with wavelength.

280nm - Tyr & Trp due to conjugation

260nm

nucleic acid impurities

Circular dichroism

Circularly polarised light beams : L and R

Differential absorption of L & R , done at diff wavelengths.

Secondary structures identified - % beta-sheet for eg.

Fluorescence Spectrometry

Shorter wavelgth = I 0

Longer wavelngth emitted = I

Eg. Trp : Abs = 280nm Emis = 340nm

X-ray crystallography

Pure crystal needed of sample. -RLS

X-ray diffraction pattern --> Electron density map --> Protein model

Crystal acts as..

a diffraction grating

space group = lattice units

Why are X-rays chosen?

Their small wavelength

NMR

nuclear magnetic resonance

NMR (low energy)

Certain nuclei perturbed by magnetic field and align either parallel or antiparallel to magnetic field. Measure disturbanace. Closer atoms have more effect on one another. Smaller structures.

Cryogenic Electron Microscopy

ELECTRON BEAMS

Focused by magnetic lens and passed through sample to then detectors. No crystals needed.

How is the protein prepared fro cryo-EM?

Purified protein is frozen on cooper plate.

What information on the protein is obtained?

Secondary structures. Better at larger structures. Simple technique that is improving in precision.

Myoglobin

An oxygen-storing, pigmented protein in muscle cells. One domain. Binds O2 tightly.

Haemoglobin

4 domains : 2 alpha, 2 beta

a coordinating His that allows iron(II) to fit in ring

Haemoglobin conformational states

R = Relaxed = Oxyhaemoglobin

T = Tense = Deoxyhaemoglobin.

Bohr effect

CO2 released, increase pH

Shift left. O2 released.

Serine proteases

Enzymes that cleave peptide bonds using serine residue as the attacking group.

Serine proteases classes

1) Chymotrypsin

2) Trypsin

3) Elastase

Chymotrypsin

Cleaves after aromatic and long hydrophobic stretches. Has the CATALYTIC TRIAD in its active site.

Catalytic triad

Ser, His, Asp

attack Ser OH, oxyanion intermediate, reaction proceeds more easily

Trypsin

Cleaves after Arg, Lys.

Asp in specificity pocket.

Elastase

Cleaves after small.

Neutrophils secrete to cross elastin layer into lungs when infection occurs - emphysema if not restored.