Isolation and Analysis of Biomacromolecules doc. Ing. Petra Lipovová, PhD.

http://biomikro.vscht.cz/vyuka/?Predmet=ibe • 7.10. 2015 Introduction - source of proteins, lysis, filtration, precipitation • 14.10.2015 Centrifugation, isolation of nucleic acid • 21.10.2015 Chromatographic techniques • 4.11.2015 Lecture canceled • 11.11.2015 Affinity chromatography • 18.11.2015 Recombination proteins • 25.11.2015 Work with program ProtNlab • 2.12.2015 Electrophoretic methods • 9.12.2015 Immunomethods • 16.12.2015 1st term of exam

Introduction source of proteins lysis of cells membrane process precipitation

1. to find suitable source of protein 2. lysis of cells 3. purification

Basic isolation steps

The source of proteins

microorganisms animal tissue plant tissue body fluid

Microorganisms

Advantages: Bacteria, yeast, fungi, algea

can be easily obtained in sufficient quantity (cultivation in fermentor...)

o selection of mutants with the required properties thermophilic organisms (production of enzymes with higher thermal stability)

o psychrophilic organisms gene engineering

Animal sources • Laboratory animals- mice, rats, rabbits • Human-body fluid (blood - plasmin, thrombin; urine -urokinase...)

• Invertebrates- insects, snails, molluscs

• Tissue culture - CHO

Plant Spinach, beets, peas, Arabidopsis thaliana, tobacco Disadvantage: the problematic growth under defined conditions plant suspension cultures

Caenorhabditis elegans

•ATCC (American Type Culture Collection) http://www.lgcpromochem-atcc.com/ •CCM (Czech Collection of Microorganisms) www.sci.muni.cz/ccm/ •FDA (Food and Drug Administration) (2001), Partial list of microorganisms and microbial-derived ingredients that are used in foods. www.cfsan.fda.gov/~dms/opa-micr.html •AMFEP (The Association of Manufacturers and Formulators of Enzyme Products) www.amfep.org • addgene.com

Where to get information about potential sources?

Your task is to isolate urease

Urease (EC 3.5.1.5) (NH2)2CO + H2O → CO2 + 2NH3

Helicobacter Pylori Urease drawn from PDB 1E9Z

http://www.madrimasd.org/blogs/microbiologia/wp-content/blogs.dir/110/files/1431/o_helicobacter.jpg

ATCC (American Type Culture Collection) http://www.lgcpromochem-atcc.com/

Plant Seed

ATCC® Number:

75043™ Price: £190.00; €285.00

Organism: Arabidopsis thaliana Heinhold

Designations:

S8-1-2A

Depositors: D Dennis, Y Poirier, CR Somerville

Biosafety Level:

1 Shipped:

dried, package of 25 seeds each order

Permits/Forms:

In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location

Seeds of Arabidopsis thaliana

Human hexokinase

brain/CNS neuroblastoma cell line

lung, small cell carcinoma

leukocyte

Homogenization

Mechanicaly: grinding, mixing, cutting, mortar with sand

Separation of cells

• centrifugation (6000 g, 10 min, 4°C) • filtration

Distribution of proteins

• Intracellular • periplasmatic • Extracellular

cytoplasmatic

periplasmatic

extracellular

cell

• Membrane proteins

Cell lysis

Methods • physical • chemical • enzymatic

Physical methods of lysis

• Mortar + glass beads, silica sand. • Alternating freezing and thawing • X-press (freezing and thawing)

X-press

piston

slit

• Douncer´s homogenizator

• Mini-BeatBeater – balotins – various dimensions

•French press - pass liquid suspension through small hole under high pressure (136 MPa)

• One Shot Disruptor

•Ultrasound - intezity - over 50 W/cm2 – cavitation

Chemical methods of lysis • osmotic lysis (erythrocyte)-suspension in hypotonic condition • change the gas pressure - nitrogen

Wikipedia

•detergents - compounds that lower the surface tension (or interfacial tension) between two liquids or between a liquid and a solid

CMC – critical micelle concentration C – concentration of detergent

•http://www.science.co.il/Biomedical/detergent_selection_table.pdf

critical micelle concentration (CMC) - the concentration of surfactants above which micelles form and all additional surfactants added to the system go to micelles.

•Y-PER Reagent significantly disrupts yeast cell wall and plasma membrane. Cells of Saccharomyces cerevisiae stain DY150 after lysis with Y-PER Yeast Protein Extraction Reagent. Arrows indicate disruption of cell wall, resulting in cell lysis.

•These images show a Streptococcus pyogenes cell experiencing lysis after exposure to the enzyme PlyC. (Credit: Daniel Nelson/UMD)

detergents •nonionic

•ionic •anionic

•kationic

•amfoteric

Detergent CMC mM

MMW Da

dialysis aplication

ANIONIC

SDS(dodecylsulfát sodný) 8,3 288,4 denaturation of proteins, for DNA, PAGE

DOC(deoxycholát sodný) 1-4 416,6 solubilization of membrane protein

N-lauroylsarkosin 7 488 solubilization of membrane protein

CATIONIC

CTAB (hexadecyltrimethyl amonium bromide)

4-5 337 ??? solubilization of membrane protein, complex with DNA, remove of polysaccharides

NONIONIC

Triton X-100 [octylphenolpoly(ethylenglycolether)n]

0,2 647 n=10

solubilizace proteinů

Tween 20 [poly(oxyethylene)n sorbitan-monolaurate]

0,06 imunobloty, ELISA

AMFOTERIC

CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)

4 614,9 solubilizace membránových proteinů

• Lysozyme – from egg whites

hydrolyse of 1,4-β-glycosidicbond between NAG a NAM

Yeast •zymolyase, lyticase – β-1,3-glucanase activity

Enzymatic methods of lysis Bacteria

Plant cells •cellulase, pectinase

Inhibitors of protease

Membrane process - filtration

•defined pore size = "cut off" (mm, Da), gravity or inert gas

microporous anizotropic

Produkt použití polyvinylidenfluorid PVDF low biding of protein polyethersulfon PES fast flow, low biding of protein esters of cellulose MCE filtration of buffers and particles polycarbonates smooth surface nylon and fiber mesh broad chemical compactibility, range 3

- 10 pH glass and a silica fibers coars filtration, solution with a high

number of particles teflon hydrofobic, org. solvents, agresiv

solutions

PVDF

0,1 - 5 μm

esters of cellulose nylon

0,22 - 180 μm 0,025 - 8 μm 0,7 – 3 μm

glass and a silica fibers

characteristic of membranes •dividing range – NMWL, MWCO, NCO •working temperatures, pressures and pH •resistance to chemicals

Membrane: Durapore Membrane Filters - PVDF (Polyvinylidene fluoride) Features: Low Protein Binding Specifications:

Pore sizes: 0.1, 0.22, 0.45, 0.65, 5 μm

Color: White

Surface: Plain

Wettability: Hydrophilic, hydrophobic

Thickness: 125 μm

Sterilization: by autoclave (121°C at 1 bar), EO or gamma

Operating temperature: 85°C max.

Gravimetric extractables: <0.5%

Protein binding capacity: 4 μg/cm²

•separation of coloidic or unsoluble particles (microorganisms) •0,05-10 µm •low pressure

Microfiltration

N2 pojistný ventil

míchadlo

retentát

permeát

membrána

•separation of proteins, virus •fractionation of proteins •size of pores 3 nm - 0,1 μm •pressure > 10 bar (145 psi, 1 MPa)

Ultrafiltration

Použití •desalting of whey •concentration

Nanofiltration

•separation of melecules with low mol. weight •size of pores 1 nm - 10 nm ∼ 200 – 15000 g/mol

- diffusion of low molecular weight substances

Dialysis

•http://www.inmed.cz/index.php?page=princip_dialyzy

Principle of hemodialysis

dialysis of 100 ml 5 M NaCl agains 1 litr of water

Precipitation

Fractionation of biomacromolecules on the basis of differences in solubility

• denaturation x precipitation

Franz Hofmeister (1850-1922)

for anions: SO42- > F- > IO3

- > NO2- >Br- > NO3

- > I- > CNS-

for cations: Li+ > Na+ > K+ > NH4

+ > Rb+ > Cs+

salting in

salting out

Ammonium sulfate precipitation

• very water-soluble • cheap • stabilization effect on proteins

The initial and final concentration of ammonium sulfate, expressed as percent saturation, are on the vertical and horizontal scales, respectively. The point of intersection of two lines drawn from these points indicates the number of grams of salt to be added to each littler of solution at the initial concentration to lead to the final concentration required.

Solid ammonium sulfate (grams) to be add to the 1 liter of solution.

G = wsat · (c2 - c1)/(csat - (Vspec/1000 ·132,14 · csat · c2))

temperature Solubility (NH4)2SO4

0 °C 70,21 g/100 ml 20 °C 75,16 g/100 ml 40 °C 80,83 g/100 ml 60 °C 87,21 g/100 ml 80 °C 94,30 g/100 ml

•G the weight of Ammonium Sulfate to add in grams per liter •wsat is the number of grams/Liter in a saturated solution of Ammonium Sulfate •c2 the molarity you want to achieve •c1 the starting molarity •csat is the molarity of a saturated solution of Ammonium Sulfate •Vspec is the specific volume of Ammonium Sulfate, which is the amount of volume 1g will add to an aqueous solution, which is about 0.54 ml

http://www.encorbio.com/protocols/AM-SO4.htm

Example: After the lysis of the cells, you have acquired a 20 ml of the extract of the protein (concentration of 0.5 mg/ml). You have choosen the ammonium sulphate precipitation (Mr 132,14) as a first step of purification of hexokinase. You find that hexokinase precipitate in 50% of saturation. That's why you have decided to do a two-step precipitation (40%, 50%). How would you follow (specific analysis and procedure)?

If you want to prevent a local increase of concentration, you will add the solution of ammonium sulphate. Count how many ml of 5 M ammonium sulfate, you must add to the 40 ml of the extract to the resulting concentrations were 0, 8 M.

mlVVV

VcVc

6,7)04,0.(8,0.5

..

1

11

2211

=+=

=

Precipitationwith organic solvents

• water-miscible organic solvents • low temperature (< 0 °C)!!!

Precipitation with acetonem (< 1µg/ml) add 5 volume of child (– 20°C) aceton and vortex 30 min at -20 °C. centrifugaton 5 min, 6,000 – 10,000 g. discard the supernatant and let the pellet air dry resuspend the pellet in buffer

aceton, EtOH, isopropanol, MetOH, propanol, diethyleter

Precipitation with organic polymers and organic compound

• PEG 4000 – 6000 – water soluble polymer, can be combined with ammonium sulphate precipitation

• tichloracetic acid – nucleic acid > 20 baze, proteins - prepare of samples for SDS-electrophoresis

Heat precipitation Relies on the differential resistance of proteins to denaturation and precipitation by heat. Perfect for the isolation of heat stable proteins

0102030405060708090

100

teplota

%

Bílkovina 1Bílkovina 2

Increase the temperature so that contaminating proteins are denatured while the desired protein remains stable and soluble.

The influence of pH on precipitation •Many proteins are precipitated from the solution in the pH that corresponds to pI •It is good to know the pI of the protein (chromatofokusace, isoelektrická fokusace)

Imunoprecipitation •antigens may be precipitated using the corresponding antibodies

•antibodies can be precipitated using the corresponding antigens

•antigens may be precipitated using insoluble form of antibody