Class 6

How Proteins Are Studied / Isolating Cells and Growing Them in Culture

protein purification first step

is to disrupt tissues and cells in a controlled fashion

homogenization

breaking open cells and tissues

using gentle mechanical procedures, called _____, the plasma membrane of cells can be ruptured so that the cell contents are released

• break apart cells with high frequency ultrasound

• use mild detergent to make holes in the plasma membrane

• force cells through a small hole using pressure

• shear cells between a clos-fitting rotating plunger and the thick walls of a glass vessel

the resulting thick soup is called homogenate

when carefully conducted, ____ leaves most of the membrane-enclosed organelles intact

homogenate or extract

resulting thick soup resulting from homogenization

contains large and small molecules from the cytosol, such as enzymes, ribosomes, and metabolites, as well as all of the membrane-enclosed organelles

fraction

cells can be separated into their component ___s

depending on which protein you are interested in, you will continue with a particular ___

cell fractioning techniques

1. low speed centrifugation of cell homogenate

2. medium speed centrifugation of previous supernatant

3. high speed centrifugation of previous supernatant

4. very high speed centrifugation of previous supernatant

low speed centrifugation

pellet contains

• whole cells

• nuclei

• cytoskeletons

medium speed cetrifugation

pellet contains

• mitochondria

• lysosomes

• peroxisomes

high speed centrifugation

pellet contains

• microsomes→ small vesicles derived from ER after cell homogenization

• small vesicles

proteins differ in

• size

• shape

• charge

• hydrophobicity

• their affinity for other molecules

these properties can be used to separate them from one another

protein separation techniques

chromatography

antibodies

electrophoresis

column chromatography

sample applied

solvent continuously applied to the top of column from a large reservoir of solvent

fractionated molecules eluted and collected

ion exchange chromatorgraphy

positively charged beads

negatively charged bound molecule

free positively charged molecule

downwards solvent flow

gel filtration chromatography

downwards solvent flow

porous bead

small molecules delayed through pore

undelayed larger molecules flow more easily

affinity chromatography

downwards solvent flow

bead with covalently attached substrate or antibody or protein that protein of interest binds to

bound enzyme molecule

also can be used to isolate binding partners of a protein of interest

• protein of interest → X

• protein X is covalently attached to matrix of a chromatohraphy column

• an extract containing a mixture of proteins is then loaded onto the column

• those proteins that associate with protein X inside the cell will usually bind to it on the column

• proteins not bound to the column pass right through, and the proteins that are bound tightly to protein X can then be released by changing the pH or ironic composition of the washing solution

immunoprecipitation

mixture of molecules

molecule of interest→ A

add specific anti-A antibodies

collect aggregates of A molecules and anti-A antibodies by centrifugation

immunoaffinity colum chromatography

column packed with beads coated with anti-A antibodies

mixture of molecules added to column from the top

molecule A (molecules of interest) binds to column with anti-A antibodies

discard flowthrough with other molecules

elute antigen A from beads

collect pure antigen A

gel electrophoresis

separates molecules based on shape, size, and charge

SDS and Beta-mercap

SDS

sodium dodecyl sulfate

detergent that helps unfold proteins and gives them negative charge

beta mercap

reducing agent that breaks disulfide bonds and helps unfold proteins

SDS and beta mercap

prepare proteins for SDS PAGE

SDS-PAGE

separates proteins based on size because

• size:charge ratios will be same for similarly charged molecules

• shapes will be same for similarly sized molecules because the proteins have been unfolded

sodium dodecyl sulfate gel electrophoresis

two dimensional gel electrophoresis

provides greater protein separation

combine two separation methods to resolve a complex mixture→ can resolve mixture of more than 1000 proteins

steps

1. use isoelectric focusing to separate proteins based on charge. Proteins electrophoresed in a narrow tube of polyacrylamide gel in which pH gradient is established with buffers. Each protein moves to a point in pH gradient that corresponds to its isoelectric point→ pH where protein has no net charge and will not move in electric field.

2. gel placed on top of gel slab; proteins subjected to SDS PAGE in direction perpendicular to that used in step 1.

3. each protein migrates to form a discrete spot

once protein is purified

break into smaller pieces using selective proteases that only cleave at carboxyl end of particular amino acid residues

use Edman degradation to determine amino acid sequence

overlap sequences and piece together overall amino acid sequence

faster way is to use mass spectrometry to determine exact mass of every peptide and identify it from a database that contains a list of every protein thought to be encoded by genome of organism protein is from

mass spectrometry

can be used to identify proteins by determining precise masses of peptides derived from them

• protein of interest excised from a polyacrylamide gel after two-dimensional electrophoresis

• then digested with trypsin

• peptide fragments loaded into mass spectrometer, and their exact masses are measured

• genome sequence databases then researched to find protein encoded by organism in question whose profile matches this peptide fingerprint

• mixtures of proteins can be analyzed with tandem mass spectrometry

protein structure determination

X-ray crystallography

NMR spectroscopy

cryo-electron microscopy

artificial intelligence prediction

X-ray crystallography

first coax purified protein into crystalline form

then mathematical computational analysis converts diffraction patterns into three-dimensional conformation of protein

NMR spectroscopy

can be used on proteins <50,000 daltons in aqueous solution

chief advantage is that protein does not need to be in crystalline form

cryo-electron microscopy

large molecular machines and integral membrane proteins often hard to crystallize, so ___ ___ __ can be used

droplet of pure protein in water placed on EM grid and plunged in vat of liquid ethane at -180 degrees C

freezes proteins in thin film of ice and rapid freezing time prevents water molecules from forming ice crystals which damage protein shape

analyzed using TEM and mathematical computational methods

resolving power of ____ approaches that of X-ray crystallography and both techniques can be used in two-pronged approach to deduce structure

artificial intelligence

uses software programs which train themselves on databases of experimentally solved protein structures

isolating and growing cells in culture

dissociate cells from tissues and separate them according to type such that have a homogeneous population of cells that can be analyzed directly or grown in culture thereby increasing cell numbers

isolate cells from tissues

1. Disrupt ECM and cell-cell junctions holding cells together

2. Gently agitate to tease apart tissue and single-cells

ECM and cell-cell junctions

hold cells together

disrupted by treatment with

• proteolytic enzymes such as trypsin and collagenase to digest proteins in ECM
  agents such as EDTA that chelate the Ca2+ on which cell-cell adhesion depends

they die eventually

drawback of cell cultures obtained from disrupting tissues is that ___ ___ ___

replicative cell senescence

replicative cell senescence

most cells stop dividing after a finite number of divisions in culture→ called ___ ___ ___

e.g.

• normal human fibroblasts typically divide only 25-40 times because of progressive shortening and uncapping of telomeres, repetitive DNA sequences that cap ends of each chromosome, because human somatic cells have turned off production of enzyme telomerase

• but human fibroblasts can often be coaxed to divide indefinitely by providing a gene that encodes catalytic subunit of telomerase and propagated as “immortalized cell line”

culture shock

protective mechanism that makes a cell stop dividing even though telomeres remain long

some human cells cannot be tricked/coaxed to divide indefinitely by providing a gene that encodes catalytic subunit of telomerase and propagated as “immortalized cell line”

to make these cells immortal→ need to activate protective mechanisms and introduce certain cancer-promoting oncogenes

rodent cells

most ___ ___ do not turn off telomerase production and therefore their telomeres do not shorten with each division

so if can avoid culture shock, these __ __ lines will keep dividing in culture

transformed cell lines

often cell lines can easily be generated from cancer cells

these cultures referred to as __ ___ __ differ from cultures from normal cells

• __ ___ __ often grow without attaching to a surface

• can proliferate to a much higher density (cells/mL) in a culture dish

tumor-inducing virus or chemical

properties of transformed cell lines can be induced in normal cell lines by transforming them with ___ ___ ___ or ___

transformed and untransformed cell lines

can be stored in liquid nitrogen -196 degrees C indefinitely and retain viability when thawed

nearly always differ from normal progenitors in tissues from which they were derived

PtK1 epithelial cell line

from kangaroo rat is advantageous to use when studying mitotic apparatus because cells stay relatively flat during mitosis unlike many other cell types

hybridoma cell lines

are factories that produce monoclonal antibodies

suspension of two cell types (normal and tumor) centrifuged with a fusing agent added—> cell fusion and transformation of heterokaryons, which are then cultured→ selective medium allows only heterokaryons to survive and proliferate. These become hybrid cells which are then cloned→ clones of hybrid cells

making monoclonal antibodies

large quantities of a single type of antibody molecule can be obtained by fusing a B cell (taken from an animal cell injected with antigen A) with a tumor cell.

The resulting hybrid cell divides indefinitely and secretes Anti-A antibodies of a single (monoclonal) types

antibodies

can also be obtained from animals but the blood will contain a variety

__ can be made in the laboratory by injecting an animal with antigen A

repeated injections of the same antigen at intervals of several weeks stimulate specific B cells to secrete large amounts of anti-A antibodies into the bloodstream

because many different B cells are stimulated by antigen A, the blood will contain a variety of anti-A antibodies, each of which binds A in a slightly different way