Recombinant ProteinProduction and Crystallography · 2018-06-05 · • Fast Protein Liquid...

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Modern Techniques in Life Science Lecture

Recombinant Protein Production and

Crystallography

June 4, 2018

Prof. Dr. Eva WolfDr. Martin Möckel

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Part I – Recombinant Protein Production - Outline

Martin Mö[email protected]

1 - Rationale behind recombinant proteins – what do we need them for?

2 - Protein expression systems / organisms• Overview• Escherichia coli• Insect cells

3 - How to access recombinant proteins – cell lysis and clearing techniques

4 - Isolation of recombinant proteins – chromatography techniques • Principle of column chromatography• Fast Protein Liquid Chromatography (FPLC)• Mainstream column-chromatographic methods

5 - Quality control of purified proteins

typicalworkflow

for proteinproduction

process

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1 - Rationale behind recombinant proteins – what do we need them for?


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1- Rationale behind recombinant proteins


Basic research: recapitulate in vivo findings -> generating direct proof for actions/interactions of

specific proteins („reconstitution“ of certain in vivo situations) study biophysical characteristics and structure (see 2nd part of the lecture) as target for antibody production laboratory routine (enzymatic reactions, e.g. for molecular cloning) etc.

Pharmaceutical industry: vaccines insulin therapeutic antibodies many more

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2 - Protein expression systems / organisms


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2 - Overview of different expression systems


Characteristics E. coli Yeast Insect cells Mammalian cells

Cell growth (time/division) rapid (30 min) rapid (90 min) slow (18-24 h) slow (24 h)

Complexity of growth medium minimum minimum complex complex

Cost of growth medium low low high high

Expression level high low - high low - high low - moderate

Extracellular expression secretion to periplasmsecretion to

mediumsecretion to

mediumsecretion to

medium

Protein folding refolding often requiredrefolding may be required proper folding proper folding

N-linked glycosylation none high mannose simple, no sialic acid complex

O-linked glycosylation no yes yes yesPhosphorylation (no) yes yes yes

Acetylation no yes yes yesAcylation no yes yes yes

gamma-Carboxylation no no no yes

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2 - Escherichia coli


Escherichia coli is the most common expression system among prokaryotes.

Advantages: fast growth easy transgene introduction heavy overexpression / large yields cheap

Disadvantages: size limit of recombinant proteins app. 100 kDa lack of eukaryotic folding machinery -> might lead

to misfolded/ aggregated overexpressed eukaryoticproteins

lack of eukaryotic posttranslational modificationsmight impact on functionality of recombinanteukaryotic proteins

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2 - Escherichia coli - expression vectors


Transgenes are introduced andmaintained using plasmids that mediate antibiotic resistance and harbour theirown origin of replication.

Transgene expression is driven by a strong promoter in most cases. Often, transgene expression is controled by a lac-operon-like system (induced by theaddition of a stable galactose-thioglycosid, IPTG (Isopropyl-β-D-thiogalatopyranosid)

In order to retain the recombinantprotein, the transgene needs an affinitytag (see slides for affinity chromato-graphy later).

vector map derived using SnapGene®

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2 - Insect cells


Sf9 cells

Advantages: allows for the production of “difficult” eukaryotic

proteins and big protein complexes strong overexpression can be achieved most of the

times require only a 28°C incubator, no need for CO2 (in

contrast to most mammalian cell systems) can be cultivated in shaker flasks (cheaper than working

with mammalian cells)

Disadvantages: long doubling time (app. 24 h, E.coli: 20 min!) tedious preparations required before expression (several

weeks) -> parallel, instead of sequential work flow isessential (start with multiple possible constructs)!

expensive media more difficult to handle (danger of contaminations)

Cells derived from the moth Spodoptera frugiperda are themost common system for eukaryotic protein production.

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2 - Insect cells - induction of recombinant protein production


Sf9 cells

The baculovirus system can be used to deliver transgenes into cultured insect cells using recombinant virus particles.

Advantage: Baculoviruses can be used in a S1 laboratory environment (compared e.g. to lentiviral infection of mammalian cells that has to be performed in a S2 lab)

Virion of AcMNPV/ baculovirus:

infection

natural occuring baculoviruses: infect subset of insects infected insects disintegrate and contaminate

leaves/plants -> larvae that feed on contaminatedplants are infected

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2 - Insect cells - baculovirus production


Baculoviruses can be produced by transfecting a huge plasmid, the „bacmid“, into insect cells. The recombinant bacmid is produced in E.coli. It contains all required genes to produce the

virus and can be modified, so that it also contains the desired transgene that will be expressedin infected insect cells.

After successfull transfection, virus particles are collected from the media and can beamplified in order to have enough virus to infect a sufficient amount of cells for proteinproduction.

It takes two to three weeks from cloning unil the production recombinant proteins.

picture from Invitrogens´ „Bac-to-Bac®Baculovirus Expression System“ manual

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3 - How to access recombinant proteins – cell lysis and clearing techniques


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3 - Cell lysis techniques


intracellular protein

requires cell lysisextracellular proteinsecretion-> can be retrieved fromthe media (or periplasmin the case of E.coli)

transgeniccell

separation of cells from media using centrifugation or filtration

use of the supernatant forprotein purification,disposal of cells

antibodies,many pharma-ceutical proteins

many researchand biotechproteins

use of the cell pellet forprotein purification, cells need to be lysed!

extracellularprotein

intracellularprotein

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3 - Cell lysis techniques


Chemical lysis: enzymatic digestion of cell wall detergents

Osmotic shock

Freeze-thaw cycles

Physical lysis: french press microfluidizer douncer homogenizer sonication

Strong sonication can heat the sample, which mightlead to the unfolding and precipitation of proteins.

Ultrasonifier

Douncer:

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4 - Isolation of recombinant proteins –chromatography techniques


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4 - Isolation of recombinant proteins


absorptive

non-absorptive

red: negatively-charged surfaceblue: positively-charged surface

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4 - 1: Principle of liquid column chromatography


Total column volume = volume of mobile phase +

volume of stationary phase

Solid/stationaryphase

Liquid/mobilephase

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4 - 2: Fast Protein (Performance) Liquid Chromatography (FPLC)


An FPLC is excellent to streamline multi-step purifications and it allows moresophisticated purification/column setups.

This makes purifications more reproducibleand reduces the hands-on time.

Modern times protein purification is almostunimaginable without FPLC machines!

columns can be exchanged

proteinsabsorb at 280 nm

chromatogram: Sironi et al., EMBO Journal, 2001, DOI 10.1093/emboj/20.22.6371

pictures: GE-lifesciences and lifeserv.bgu.ac.il

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4 - 3: column-chromatographic methods


affinity,(ion exchange)

ion exchange,hydrophobic interaction

gel filtration

Multi-step purification setups are superior to single-step setups, as they allow for higherpurity (and often a more reproducible activity profile) of recombinant proteins:

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4 - 3: column-chromatographic methods - affinity


Bind-elute principlewith specific bindingand elution conditions:

Eluent competes withprotein for ligand binding:

protein-concentratingeffect

Affinity chromatography is a very selectivetechnique, therefore often used as a firststep to enrich recombinant proteins fromcell lysates. Most of the times, the proteinof interest needs to be equipped with an artificially introduced affinity tag.

recombinant fusion protein

high eluent concentration in bufferlow eluent concentration in buffer

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4 - 3: column-chromatographic methods - affinity


Eluent

There are various artificial affinity tags available, some examples are:

recombinant fusion protein

specific protease cleavage site allowsaffinity tag removal (while protein isbound to column or after elution)

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4 - 3: column-chromatographic methods - ion exchange


Proteins are amphoteric:

Ion exchange chromatography (IEX) separates molecules on the basis of differences in their net surface charge at a certain pH

Bind-elute principle:

binding at low ionic strength(and correct pH range) andelution using high ionicstrength (high salt) conditionsor adjusting the pH to theisoelectric point of the protein:

high [salt]

low [salt]

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4 - 3: column-chromatographic methods - size exclusion


Size exclusion chromatography (SEC) separates molecules on the basis of theirsize (and shape). The matrix is inert (non-absorptive technique). Compatible with various buffers Ideal final polishing step Problem: dilution of the sample

porous beads: small molecules enter thepores, while larger molecules do not/less:

stationaryphase

mobile phase

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5 - Quality control of purified proteins


www.imb.deMartin Mö[email protected]

5 - Quality control of purified proteinsSDS-PAGE:

staining of proteins with coomassie brilliantblue, molecular weight validation andidentification of impurities (other proteins)

Analytical size exclusion chromatography:

identification of proteinaggregates/oligomers

protein complexes: (correct) stoichiometry

Spectroscopy/spectrometry:

Ultraviolet (UV) spectroscopy : identification of contaminating nucleic acids

Infrared (IR) spectroscopy: secondary structure of protein

Mass spectrometry: identity of correct protein sequence/length,

post-translational modifications

www.imb.deMartin Mö[email protected]

1 – Capture of His-tagged protein from E. coli lysate using a Ni-functionalized column (affinity):

3 – Polish and homogeneity check using size exclusion:

Exemplary purification flow

2 – Intermediate ion exchange step/concentration:

MacromolecularX-ray crystallography

Eva WolfStructural Chronobiology

JGU and IMB Mainz

X-ray crystallography

Why X-rays ?

Why crystals and crystallography ?

Abbe limit: resolution limited bywave length of used „light“ source→ use X-rays with l about 1 Å

- Enhance X-ray diffraction signal by regular repetition(signal : noise)

- Radiation damage

Intensity Ihkl ~ number of unit cells

Dimensions of biological Structures

Atoms Small Macromolecule Particle Organelle, Eucaryotic

molecule Protein/DNA/RNA Bacteria Cells

C-C- Ribose Domain Ribosome Mitochondria Erythrocyte

bond (~ 4 Å) (2.5 - 5 nm) (~ 30 nm) Bacteria

1.54 Å Virus (1-2 mm) (5-8 mm)

(10-100 nm)

0.1nm 1nm 10nm 100nm 1mm 10mM

1 Å

Dimensions biological Structures - Suitable techniques for visualization

Atoms Small Macromolecule Particle Organelle, Eucaryotic

molecule Protein/DNA/RNA Bacteria Cells

C-C- Ribose Domain Ribosome

bond (~ 4 Å) (2.5 - 5 nm) (~ 30 nm)

1.54 Å Virus (10-100 nm) (1-2 mm) (5-8 mm)

0.1nm 1nm 10nm 100nm 1mm 10mM

1 Å

Light microscopy

Resolution ~ l/2, > 250 nm

Superresolution lightmicroscopy: ~ 10-20 nm

Atomic resolution

(~ 1 - 3 Å) :

X-ray crystallographyl x-ray ca. 1 Å

NMR

Single particle EMl 100 keV e- : 0.037 Å

Electron Microscopy

Single particle

10-20 Å negative stain

(e.g. Uranylacetate)

> 3 Å Cryo-EM

(direct detector, motion

correction, sample) Electron MicroscopyCell biology

X-ray crystallography

NMR (nuclear magnetic resonance)

Single particle EM

Modelling

Ab Initio vsHomologie

Crystal, broad MW range, high resolution (typically up to1.5 Å)

Solution, dynamics,MW typically < 30 kDa

MW > 200 kDa

resolution typically up to 3 Å

Resolution6 Å

3.0 Å

4.0 Å

1.8 Å

1.0 Å

10 Å – cryo-EM

Ring of

6 subunits,

hexamer

0.5 Å

Macromolecular X-ray crystallography

mg

Cu Ka = 1.54 Å Variable l, usually around 1 Å

FT

F(hkl) = |F| * eia

In vitro =„in glass“

SDS-PAGE

Rotating anode Synchrotron

„PhaseProblem“

X-ray diffraction pattern Electron density

Protein Expression (recombinant) and Purification

Crystallization

X-ray data collection

Structure determination

Phasing stategies, Electron density calculation

Model building and Refinement


Structure Validation and Evaluation (Geometry)

mg of pure, homogeneous protein: Purity: SDS-PAGE > 95% pure.Non-aggregated, stable, folded,single conformational andoligomeric state: SEC, MALS/SLS, DLS, CD, SAXS

Crystallisation of Macromolecules

Multiparameter search for „right“ conditions

- pH

- Precipitant : PEG (Polyethylenglykol), MPD (2-Methyl-2,4-Pentanediol), Ethanol, Isopropanol, Salt (e.g. AmSO4, LiSO4, NaCl, MgCl2, NaOAc)

- Ionic strength

- Additive (chemical helping crystallization)

- Temperature (4°C, 20°C)

- Protein construct (Fragment, Organism, Ligands)

- Protein/Ligand: Concentration , Stoichiometry

Screens to find good conditions.

Crystallization robots and imaging systems.

Optimization of Protein Crystals

Spherulites Clusters of needles

Single needles („1D“)Plates („2D“)

3D!

< < <

< <

best

Crystallisation by Vapour Diffusion

„Hanging drop“

Crystallization plates:

96 well 24 well

Typical drop volumes:

1µl + 1µl (24 well, manual)

100 nl + 100 nl (96 well, robot)

H2OReservoir:70 µl (96 well)0.5 - 1ml (24 well)

„Sitting drop“

Crystallisation by Vapour diffusion: Phase diagram

Hangingdrop

H2O

Drop: Reservoir + Protein

Red arrows: concentration over time, developmentduring crystallisation. (minutes to weeks)

Undersaturated

Supersaturated

A typical screen result – 96 well

Lots of precipitate in different conditions …..

Hopefully at least one

drop with a crystal …

If you don‘t get a crystal.. : Change theprotein construct

Lots of precipitate in different conditions …..

Design crystallizing proteins by limited proteolysis

212

118

66

43

29

20

14

M Chymotrypsin GluC

Identify flexible protein regionsthat are accessible and cleavedby proteases.

Mass spec to determine domainboundaries of stable degradationproducts. Reclone, express, purify.

Or change the organism, e.g. → →

Remove flexible parts→ better crystal packing

Design crystallizing proteins by limited proteolysis –improve crystal packing

Quite well packedalready


Crystallization


Phasing, Electron density calculation




General Setup

X-ray source

Optics:Mirrors,Monochromator Crystal

Detector

Crystal freezing and Mounting

Collect data at 100K (reduce radiation damage)

- define cryogenic solution (e.g. add glycerol)

- mount crystal in loops

- freeze crystals in liquid nitrogen

- nitrogen gas at data collection

0°C = 273,15 K 100 K = -173.15°C

Liquid nitrogen evaporates at 77 K (-196°C) at normal pressure.

X-ray sources in Macromolecular Crystallography

Rotating Cu-Anode („home source“):

characteristic monochromatic Cu-Ka-

radiation (1.54Å)

Detector: Image plate

Rotate crystal at data collection

Synchrotron sources for macromolecular CrystallographyDESY-PETRA III

(Hamburg)

Synchrotron: high brilliance & variable wave length

→ electrons travel in a ring accelerated perpendicular to magnetic field

→ tangentially emitted Synchrotron radiation

→ high brilliance (high intensity, low divergence), variable wave length

→ smaller crystals, larger unit cells (larger 3D-structures), better data quality, experimental phasing with anomalous signals, time resolved studies

Experimental setup at Synchrottron (ESRF)

Detector:

CCD

(fast)

Cryo stream, nitrogen gas

crystal

Mount crystals manually: potentially safer, but takes longer and can be hard at 3 am at night

Automatic sample changer – faster, not always reliable, but less work

Automatic sample changer at work – the scientist stays outside ..

Pilatus: large area pixel detector

Reward: High resolution 3D–structures of

multi-subunit macromolecular complexes

Large/few unit cells, potentially small crystals → weak signal→ need high brilliance synchtotron radiation and sensitive detectors

E.g.- Ribosome: Protein biosynthesis- Nucleosome: DNA-packaging, Chromatin- RNA-Polymerase II : Transcription

Ribosome (1.6 MDa) Nukleosome (206 kDa) RNA-Polymerase II,

~ 500 kDa

Single particle Cryo-EM - recent developments

Direct electron detector: electrons detected directly

- more sensitive and much faster than CCD cameras→ record movies (many frames per second)

New image-processing tools: correct images for beam-induced

sample movements → movie processing and motion correction

→ Resolution up to 3 - 4 Å for larger complexes

Advantage: don‘t need crystal

Draw back: - very expensive equipement- sample preparation tedious- resolution not beyond 3 A

(X-ray still higher resolution)- not very suitable for < 200 kDa

Examples for high resolution Cryo-EM structures

Ion channel, ≈3 Å Hydrogenase, ≈ 3 Å

Mitochondrial ribosome, ≈ 3 Å g-Secretase (170 kDa) ≈ 4.5 Å (Nature 2015: 3.4 Å)


Crystallization






Steps to get a crystal structure – structure determination, model building

mg

Cu Ka = 1.54 Å Variable l, usually around 1 Å

FFT

F(hkl) = |F| * eia

„Phase Problem“

Structure determination - The Phase Problem

FFT

F(hkl) = |F| * eia

Amplitude Phase

1. Phases of similar structure:

Molecular Replacement

2. Experimental phases:

Heavy atoms or anomalous

scatterers

FFT

Light and electrons:lens as physical

Fouriertransformator

→ Microscope

X-rays: no lens, calculate

Fourietransformation (FFT)


Crystallization






???????

Interpret Electron Density

Experimental density

Skeleton of Protein

(building program)

Corrected Skeleton

Ca-backbone based on corrected skeleton

1

2

3

4

Interpret Electron density – Model building

Assign Sequence to electron density

1. Known fold/structure (molecular replacement)

→ look for expected secondary structure elements

(e.g. five-stranded antiparallel b-sheet, long a-Helix)

→ orientation known

- Primary sequence alignments

Experimental densityCa-Backbone of Protein

Interpret Electron density – Model building

2. Unknown Structure

- Identify secondary structure elements (b-Sheet, a-Helix)

- Look for Ligands/Cofactors (e.g. Nucleotide GDP)

→ define active centre

→ positive |Fobs| - |Fcalc| difference density

GDP: 2.0 Å (Mg2+ yellow, H2O red)

Parallel or antiparallel ?

parallel antiparallel

Parallel and antiparallel ß-sheet

Antiparallel !

parallel antiparallel

Parallel and antiparallel ß-sheet

2. Unknown Structure

Look for amino acids with large side chains

(Phe, Trp, Tyr)

Se-MAD/SAD: Selenium positions (Se-Methionine),

phasing by anomalous dispersion

→ positive anomalous difference density |F+| - |F-|

2 Å

Build Model

Ribbon SurfaceLigand

Refine

Interpret Electron Density


Crystallization






Evaluate Geometry of the Final Model

Main chain torsion angles Ca – N (F, Phi) and Ca – C=O (Y, Psi)

between peptide bonds → allowed values for secondary structures.

Check residues with disallowed F,Y-angle combinations

→ Ramachandran Plot

w=180°

Ca

Phi

Psi

R

92.9% allowed (red)

7.1 % additionally allowed(yellow)

No residues disallowed.

→ good geometry

Example:

Evaluate Structure : What can we learn ?

Follow-up experiments ?

IIGP1(Ghosh et al., 2004)

PDB (Protein Data Bank), entry 1TQ4

Fold / Topology:

→ Topology-Diagramm

Similar structures in pdb ?

→ DALI-Server (www)

Fold type: e.g. SCOP

(Structural Classification of Proteins)

Analyse molecule surface for potential interaction sites withother molecules: electrosatic surface potential

Example: Dimer of GTPase IIGP1 (Ghosh et al, 2004), surface charges

Red: negative, blue: positive, white: hydrophobic.

Positive

Negative

Analyse Protein-Protein-

Interactions of complexes

IIGP1-Dimer: Interfaces I and II

↓

Structure based interface mutants

Interface I (B): 800Å2 buried surface;

helical interface

Mutate: Leu44/Arg (steric),

Lys48/Ala (hole),

Lys48/Glu (charge reversal)

Interface II (C):1200Å2 buried surface;

GTPase Domains.

Mutate: Met173/Ala (hole),

Ser172/Arg (steric)

Analyse Protein-Ligand Interactions

GDP GppNHp

2Å

2,7Å

Core Facilities Lecture Protein Production Crystallography slides MMFoliennummer 1Foliennummer 2Foliennummer 3Foliennummer 4Foliennummer 5Foliennummer 6Foliennummer 7Foliennummer 8Foliennummer 9Foliennummer 10Foliennummer 11Foliennummer 12Foliennummer 13Foliennummer 14Foliennummer 15Foliennummer 16Foliennummer 17Foliennummer 18Foliennummer 19Foliennummer 20Foliennummer 21Foliennummer 22Foliennummer 23Foliennummer 24Foliennummer 25Foliennummer 26

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Recombinant ProteinProduction and Crystallography · 2018-06-05 · • Fast Protein Liquid...

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