Methods in Protein Biochemistry

anthrax

disease from bacteria, made of 3 components including protective antigen protein, which binds to receptors on surface of target cell changes it to form pore and allowing the 2 toxin proteins to enter

protective antigen is a target for vaccines and therapies

protein purification

start with isolated or tissue samples, extract proteins, isolate fractions using the biochem differences in the proteins to seperate, use biocehm assay to identify proteins of interest among all other proteins in sample

luciferase

enzyme that converts D-luciferin to oxyluciferin, the reaction also creates light, ATP-dependent, and light is the assay readout.

this is done in fireflys

preparing cell extract (protein purification)

take tissue, mince it, suspend it in isotonic buffer, the cell suspension leads to sonication, shearing or mild detergents which all go back to the cell extract

centrifugation

separates the proteins using density, centrifugal force and centrifugation time, high density go to bottom, low top

column chromatography

separate proteins using interactions with solid gel matrix, gel filtration separates based on size, ion-exchange separates based on charge and affinity separates based on ligand interactions

how the column chromatography works

have the full mixture, continue to add buffer to it, each separate protein runs through the tube

gel filtration chromatography

the large proteins are too big to enter the pores of the gel matrix beads so they bypass them completely taking the shortest path (eluted first), the smaller proteins penetrate into the bead pores taking longer time

pooled fractions typically tubes 7-9 contain the highest

ion exchange chromatography

postively charge proteins get bound to the beads (-) the negatively charged proteins collect and elute first, positive elute last

the bound proteins are washed using elution buffer with cations to displace the + proteins

affinity chromatography

the beads are coated with covalently bound ligand specific to target molecule, the nonspecfic proteins elute first since they lack the proper shape/affinity to bind to ligand, target elute last

add elution buffer packed with free competing ligands which outcompete the bead bound ones lock onto target and wash them out

PAGE- polyacrylamide gel electrophoresis

uses electric field to separate proteins in the matrix, 3 types: SDS, IEF, 2-D PAGE

SDS page

amphipathic, proteins are denatured with a strong, anionic detergent coating them with - charge migrating to anode

Separates proteins based on molecular mass (MW), smaller proteins travel faster and farther cuz slip through polyacrylamide pores

higher polyacrylamide percentage create smaller mesh pores better to separate

IEF PAGE

Polyacrylamide gel matrix contains a stable pH gradient

Separates proteins horizontally on the basis of charge as a function of pH (pI), cations go to cathode, anions to anode left with two separate sides

proteins stop where pH = pI, because the net charge is zero.

2-D PAGE

Combines 1-D IEFand 2-D SDS-PAGE

Separates proteins based on both pI then IEF strip loaded onto gel separates vertically by MW

spots can be identified by Edman or mass spec.

2-D DIGE

compares protein expression between 2 samples directly on the same gel using fluorescentes a variation of 2D-PAGE

Frederick Sanger

nobel prize for chemical cleavage method to sequence oligopeptides he determined the 51 amino acid for insulin and nobel prize for enzyme based dna sequencing

per edman

improved sangers sequencing method by using PITC to tag N-terminal amino acids

the edman method

determine amino acid sequence of peptide or protein by removing and identifying one residue at time, PITC reacts with N-terminal under basic conditions, treat with TFA to cleave first peptide bond release the N tagged residue released as PTH and identified by HPLC

leaves a new free N-terminus, allowing the cycle to be repeated sequentially to map out the full sequence.

differential protease cleavage

used to generate small overlapping polypeptides that can be sequenced by Edman degradation, each batch of fragements are sequenced independently

trypsin cleaves C terminal side of + charged residues

chymotrypsin cleaves C terminal of bulky aromatic residues

Tandem mass spectrometry

used to determine amino acid sequence of peptides by breaking it down and measuring its weight twice

1. Ionization: Charge the sample.

2. MS1: Select a specific protein piece.

3. Collision: Smash the piece into fragments.

4. MS2: Weigh the fragments.

5. Detection: match masses to predicited fragment to read sequence

solid phase peptide synthesis

builds C to N terminus, the first amino acid is anchored to solid resin bead by its carboxyl group

1 starts by deblocking removing Fmoc to expose free amino group

2 incoming amino acid is treated with DCC to activates its carboxyl carbon

3 activated incoming amino acid attacks free amino group creating new peptide bone repeated until full chain

John Kendrew

used X-ray diffraction data of protein crytal to build low resolution 3-D model of myoglobin

X-ray crystallography

force millions of the protein to line perfeclty into solid protein crystal, shoot X-ray through it to scatter the atoms and diffract it leaving pattern, computer analyzes the spots to calculate and then can build a model

NMR spectroscopy

place liquid protein sample in magnent and hit with radio waves, atoms absorb energy and emit signal back, computer maps this which tells you how close they are in space, generates ensemble showing what part of protein is rigid and flexible

X-ray simplified

needs to crystallise and diffract, solid, small to medium soluble, long screening short processing, high atomic resolution but crystallisation can change conformation of proteins due to packing interactions

NMR simplifed

soluble, in buffered solution, small polypeptide, short prep time long analysis, soluble state closer to real, more expenisve from isotopic labeling and no large proteins

Cryo-electron microscopy

flash freeze liquid protein, creates vitrified protein, put into transmission electron microscope to shoot electrons through, microscope captures projections of proteins then computer shorts the images and makes 3D model

cryo-electron simplified

flash freezes particles in vitreous ice use electron beam capture 2D image to make 3D, large macromolecular complexes but has lower resolution and smaller proteins are tricky

immunoglobulin

Fab fragment, with the variable domains of the heavy and light chains binding the antigen

polyclonal vs monoclonal antibodies

P: recognize multiple epitopes on antigen molecule

M: recognize only one epitope on antigen molecule

polyclonal vs monoclonal in mice and rabbits

Polyclonal (Rabbits): A mixture of different antibodies collected directly from animal blood serum that recognize multiple spots on an antigen.

Monoclonal (Mice): A pure line of identical antibodies created by fusing mouse B cells with tumor cells (Hybridomas) to make an immortal factory that targets one single spot.

western blotting

Tells you if a protein is present and its exact size/amount by SDS PAGE then transferring it from a gel to a membrane and using enzyme-linked antibodies to create a visible band.

immunofluoroscence

cells are fixed to preserve structure, tells you the exact visual location of a protein inside an intact cell by using antibodies linked to glowing fluorescent dyes under a microscope.

ELISA

measure exactly how much target protein is in fluid sample

capture the anitbody, add the sample, detect the antibody and watch the colour change, the darker it is the more target protein present

rapid antigen test

drop sample if SARS CoV 2 shows up it binds to loose mobile antibodies the strip has a line of fixed antibodies (green) stuck to paper and then the coloured nanoparticles get trapped and pile up giving visible line aka positive, the control line is further down paper of second antibodies that catch leftover unused mobile antibodies

specific activity calc

= total activity units ÷ total protein (mg). It measures purity

fold increase 1 step calc

= specific activity of that step ÷ specific activity of the previous step.

total fold purification calc

= specific activity of that step ÷ specific activity of the crude extract.

yield %

= total activity at that step ÷ total activity in the crude extract × 100.

differential centrifugation

increasing g-force and time gives at least four subcellular fractions, each tested with an assay.

epitope

specific site on antigen on antibody recognizes

2 properties that influence development of purification strategy

physical like number of amino acids and sequence of chains and relative abundance in specific cell type

homogenization techniques

sonication (vibrates disrupt cell membrane), shearing (force cell through small opening) and mild detergent (disrup cell)

con of sanger method

each round of cleavage needed new sample of protein to add becasue acid hydrolysis destroyed it

most difficult steps in x-ray crystallography

growing the diffraction quality crystals and determining the phase of the diffracted x-rays