Purificazione di proteine umane da animali

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Purificazione di proteine umane da animali

• Basse rese
• Difficili da purificare
• Costoso
• Possibilita di malattie

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How can we synthesise human proteins?

• Use bacterial cells
• Human gene lacks
• Bacterial promoter
• Bacterial terminator
• Bacterial ribosome binding site
• Cannot deal with introns

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Dealing with introns
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Protein Expression in E. coli
Advantages and
Disadvantages

• Post-transcriptional modification
• Post-translational modification
• Poor folding
• Proteolysis
• N-terminal Methionine
• Complicated purification
• Lack of efficient secretion
• Possible toxicity

• Inexpensive
• Easy to manipulate
• Well characterized
• Grows quickly
• rProtein up to 50 total protein

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E. coli Expression Vector
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E. coli Promoters
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Weickert, et al., 1996
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E. coli Expression Vector
E. coli Expression Vector
Promoter
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Optimizing Expression
What if expression is low?

• Examine codon usage
• Decrease message stability
• Premature termination of transcription
• Premature termination of translation
• Frameshifts, deletions, and misincorporation

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Codon Frequency in E. coli
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Optimizing Expression
What if expression is low?

• Examine codon usage
• Minimize GC at 5
• Add terminator
• Add fusion and/or tags
• Growth conditions

• Combined approach

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Expression of Fusion Proteins

• Increase expression
• Ease of purification

• Ease of detection
• Increase solubility
• Increase stability

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Examples of Fusions/Tags

• Hexahistidine-tag
• GST
• MBP
• CBP/Intein
• Arg-tag
• S-tag

• Ni affinity
• GSH
• Amylose
• Chitin
• Ion-Exchange
• RNAse

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Insoluble Proteins

• Growth Temp
• Media
• Expression rate
• Chaperones
• Coexpression of subunits
• Express as polymer
• Redox potential
• Periplasmic expression
• Fusion
• Tags
• Express as a fragment
• Denature and renature
• Combined approach

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Improving Protein Stability

• Protease inhibitors
• Protease-minus host
• Periplasmic expression
• Growth temperature
• Combined approach

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MANIPOLAZIONE DELLESPRESSIONE GENICA NEI
PROCARIOTI
-PROTEINE DI INTERESSE TERAPEUTICO E
COMMERCIALE POSSONO ESSERE PRODOTTE IN E. coli
CON TECNICHE DNA RICOMBINANTE -PROMOTORE -SEQUEN
ZE LEGANTI I RIBOSOMI ( 6-8 nt Seq. di Shine
Dalgarno) -NUMERO COPIE DEL GENE
CLONATO -LOCALIZZAZIONE FINALE PROTEINA -STABILITA
PROTEINA IN CELLULA OSPITE
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GENI IN PROCARIOTI POSSONO AVERE -ESPRESSIONE
COSTITUTIVA -ESPRESSIONE REGOLATA (es. lac
operon) NELLA PRODUZIONE DI PROTEINE ETEROLOGHE
IN BATTERI VENGONO UTILIZZATI SPESSO
PROMOTORI FORTI E REGOLABILI UNA PRODUZIONE
CONTINUA PROVOCA -INIBIZIONE FUNZIONI
CELLULA -PERDITA ENERGIA -PERDITA PLASMIDE
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Bottlenecks to efficient protein expression in E.
coli
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Inefficient transcription
No or little protein synthesized
u
Promoter choice and design
l
Inefficient translation
No or little protein synthesized
u
Codon usage Transcript stability Transcript
secondary structure
u
u
Aggregation or degradation
l
Inefficient folding (cytoplasmic or periplasmic)
u
Improper secondary, tertiary or quaternary
structure formation Inefficient or improper
disulfide bridge formation Inefficient
isomerization of peptidyl-prolyl bonds
u
u
Aggregation or degradation
l
Inefficient membrane insertion/translocation
l
Toxicity
Cell death
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Folding chaperones in de novo folding
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GroEL-GroES co-expression and low temperatures
improve leptin folding
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However, this strategy does not always work
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PROTEINE DI FUSIONE -PER EVITARE DEGRADAZIONE DI
PICCOLE PROTEINE ETEROLOGHE QUESTE VENGONO
PRODOTTE COME PROTEINE DI FUSIONE CON UNA
PROTEINA STABILE DELLORGANISMO OSPITE. -I DUE
cDNA DEVONO ESSERE FUSI MANTENENDO LA CORRETTA
CORNICE DI LETTURA
PROMOTORE REGOLABILE
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GST o MBP UTILIZZATE PER PURIFICAZIONE
SITO DI TAGLIO PER PROTEASI
PROTEINA DI INTERESSE
INDUZIONE DI ESPRESSIONE PROTEINA DI
FUSIONE (PROMOTORI REGOLABILI)
TRASFORMAZIONE IN BATTERI
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-
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pGEX
GST comes from Schistosoma mansoni
Foreign gene
GST

• IPTG
• induction
• High level
• expression

tac
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PURIFICATION OF GST FUSION PROTEINS
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PURIFICATION

• EASY
• AFFINITY CHROMATOGRAPHY

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PURIFICATIONDETAILS

• GROW SAY 1L CULTURE TO MID LOG PHASE
• ie OD260 0.4 0.7
• SPIN DOWN CELLS
• SONICATE IN PRESENCE OF PROTEASE INHIBITORS
• POUR LYSATE OVER GLUTAHIONE SEPHAROSE BEADS IN A
  COLUMN

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GLUTATHIONE SEPHAROSE
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FUSION PROTEIN
FOREIGN PEPTIDE
GST
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FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE
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PURIFICATION

• WASH COLUMN EXTENSIVELY
• ELUTE WITH REDUCED GLUTATHIONE
• RESULTS IN PURE GST FUSION PROTEIN

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COMPETITIVE ELUTION WITH GLUTATHIONE
SEPHAROSE
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RESULT OF AFFINTY PURIFICATION AND REMOVAL OF GST
MOIETY
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pQE VECTORS (Qia Express)

• Hex-histidine tag system
• Produce peptides with 6 histidines fused to N or
  C terminus
• Allows Nickel Chelate Affinity Chromatography

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pQE VECTORS (Qia Express)

• Promoter
• engineered from phage T5 lac operator
• 2 operator sites
• IPTG inducible
• Expression in host containing multiple copies of
  pREP4 which has lacI

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pQE VECTORS (Qia Express)

• Interaction between Ni2 resin called NTA is
  very strong and chemically resilient
• every Ni2 binds 2 his residues in a
  non-conformation dependent manner
• therefore resists strong denaturants eg 6M
  guanidium HCl

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pQE VECTORS (Qia Express)

• Elution
• competitive with imidazole

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pQE VECTORS (Qia Express)

• Removal of His tag?
• not necessary usually
• many hundreds of proteins purified with no effect
  on structure
• not immunogenic

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PROTEINE DI INTERESSE TERAPEUTICO IN
PROCARIOTI -RISCHIO CONTAMINAZIONE VIRALE
NULLO -RISCHIO ALLERGIE NULLO (vengono prodotte
proteine umane)
PRODUZIONE DI INSULINA UMANA IN E. coli -70
MAIALI PER 1 PAZIENTE PER UN ANNO -E. Coli NON
SA MODIFICARE premRNA EUCARIOTICI E PRODURRE
MODIFICHE POST-TRASCRIZIONALI
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SINTESI INSULINA IN CELLULA PANCREATICA
CATENA A 30 aa CATENA B
21 aa
Unite da ponti S-S
ESONE 2
ESONE 1
PREPROINSULINA
PEPTIDE SEGNALE
PROINSULINA
FORMA S-S
IN APPARATO DEL GOLGI UN ENZIMA RIMUOVE 33aa
INSULINA
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PRODUZIONE DI INSULINA RICOMBINANTE IN BATTERI
-Plasimidi separati codificano per Catena A e
B -promotore trp e alcuni codoni iniziali
trp -seq per il trp sono eliminate con
trattamento con bromuro di cianato -catene
mescolate assieme e tramite un processo chimico
si formano legami S-S
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PRODUZIONE ORMONE DELLA CRESCITA UMANO IN E. Coli
-Peptide di 191 aa -Carenza provoca nanismo -GH
da animali non è efficace sulluomo -80 ipofisi
di cadaveri umani per un paziente per un anno
(alto rischio infezioni)
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PRODUZIONE DI GH RICOMIBINATE IN BATTERI
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SALMONELLA

• Expression host
• Live vaccine delivery

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SALMONELLA

• Salmonella is itself a pathogen S.typhi causes
  typhoid
• It is possible to vaccinate aganst with
  attenuated strains
• Attenuated Salmonella can persist in the gut and
  disseminate
• Induces mucosal systemic cellular humoral
  responses
• It has potential to be engineered as one shot,
  multivalent vaccines

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SALMONELLA

• Recognises E.coli promoters and origins of
  replication
• therefore existing vectors can function
• Several ways of attenuating Salmonella have been
  discovered

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EXPRESSION SYSTEMS

• MOST USE PLASMIDS
• PROBLEMS
• INSTABILITY
• TOXICITY
• pIP-pET DUAL PLASMID
• NirB-ANAEROBIC INDUCIBLE
• BALANCED LETHAL

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pIP-pET DUAL PLASMID
pL
c1ts
T7 RNA polymerase
kanR
c1ts l repressor active 28C, inactive at
37C pL l left promoter
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pTECH VECTORS

• THESE USE THE NIRB PROMOTER
• NIRB ENCODES NADH-DEPENDENT NITRITE REDUCTASE
• NIRB INDUCED IN ANAEROBIC CONDITIONS eg GUT
  TISSUES

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pTECH VECTORS
Khan made this vector

• Oral immunisation,
• single dose in mice
• protected against
• Salmonella
• Tetanus toxin

GST
tetanus toxoid
NirB promoter
pTECH
AmpR
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BALANCED LETHAL SYSTEM

• OTHER SYSTEMS DESCRIBED CARRY ANTIBIOTIC
  RESISTANCE-UNDESIREABLE
• THESE VECTORS COMPLEMENT LETHAL DELETION IN HOST
• GENE FOR B-ASPARTATE SEMI-ALDEHYDE DEHYDROGENASE
  OR asd
• asd MUTANTS HAVE ABSOLUTE REQUIREMENT FOR
  DIAMINOPIMELIC ACID (DAP) A CONSTITUENT OF THE
  CELL WALL
• THERE IS NO DAP IN MAMMALS

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Balanced Lethal
foreign gene
trc promoter
pYA292
asd
asd complements asd D host is thus stable
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Heterologous Expression in Yeast

• Codon usage is closer to human
• Glycosylation of exported proteins
• Purification of proteins from the medium
• Ease of transformation
• Ease of growth

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EXPRESSION IN PICHIA PASTORIS
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PICHIA PASTORIS

• USES ALCOHOL OXIDASE 1 (AOX1) PROMOTER
• AOX1 IS INDUCIBLE BY METHANOL AND GENE IS
  EXPRESSED AT VERY HIGH LEVELS
• THERE ARE THREE BASIC STEPS

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STEP1

• CLONE GENE OF INTEREST INTO SHUTTLE VECTOR
  DOWNSTREAM OF AOX1 PROMOTER IN E. coli

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STEP2

• TRANSFORM HIS- PICHIA PASTORIS YEAST WITH
  PLASMID. SELECT FOR HIS STABLE INTEGRANTS
  DISRUPTED IN THE AOX1 LOCUS

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STEP2
TT
gene of interest
HIS4
AOX1 promoter
3 AOX1
P.pastoris chromosome
gene of interest
pAOX1
3 AOX1
INTEGRATION
TT
3 AOX1
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• Pichia pastoris production of single-chain
  antibody fragments (scFv)
• A CASE STUDY
• 1. PLACE scFv cDNA in vector pPIC9K

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pPIC9K
PLACE scFv cDNA in vector pPIC9K
scFv cDNA
His 6 tag
pAOX1
a-mating type secretion signal
ALL RECOMBINANT STEPS DONE IN E.coli
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scFv expression in P. pastoris

• 2. Transform HIS- P. pastoris by electroporation
• Select on minimal media
• Check medium for product after methanol
  induction.
• POSITIVE

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scFv expression in P. pastoris

• 4. Large scale up
• 5 litres capacity stirred reactor
• 4L medium plus 400 ml starter culture
• Grow 17h _at_ 30oC in glycerol
• Dense
• Keep pH stable _at_ 6.0
• Induce 48 h with methanol
• Harvest culture medium
• Adjust pH to 7.4 and Affinity Purify by Nickel
  Chelate Chromatography

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YIELDS

• For scFV antibody 250 mg per L
• OTHER EXAMPLES
• highest yield
• tetanus toxin frag C 12g per L
  (INTRACELLULAR)
• a amylase 2.5g
  per L (SECRETED)

CAN WORK ON INDUSTRIAL SCALE
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YIELDS
PRODUCT YIELD g per L
ENZYMES
Invertase 2.3
a amylase 2.5
ANTIGENS
Pertussis Antigen P60 3.0
Tetanus toxin fragment C 12.0
HIV gp120 1.25
Tick antigen 1.5
CYTOKINES
TNF 10.0
Interferon alpha 0.4
PROTEASES
Carboxypeptidase B 0.8
ANTIBODIES
Rabbit single chain Fv 0.25
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ADVANTAGES OF EXPRESSION IN P. pastoris

• EUKARYOTE- some post-translational modification
• MICRO-ORGANISM
• easy to manipulate
• cheap
• YEAST advanced molecular genetics
• HIGH YIELDS

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• Molecular Farming
• A new field where plants and animals are
  genetically engineered to produce important
  pharmaceuticals, vaccines, and other valuable
  compounds.
• Plants may possibly be used as bioreactors to
  mass-produce chemicals that can accumulate within
  the cells until they are harvested.
• Soybeans have been used to produce monoclonal
  antibodies with therapeutic value for the
  treatment of colon cancer.

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Molecular Farming

• Plants have been engineered to produce human
  antibodies against HIV
• Pharmaceuticals has begun clinical trials with
  herpes antibodies produced in plants.
• The reasons that using plants may be more
  cost-effective than bacteria
• Scale-up involves just planting seeds.
• Proteins are produced in high quantity.
• Foreign proteins will be biologically active.
• Foreign proteins stored in seeds are very stable.
  
• Contaminating pathogens are not likely to be
  present.

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Molecular Farming

• Edible Vaccines
• People in developing countries have limited
  access to many vaccines.
• Making plants that produce vaccines may be useful
  for places where refrigeration is limited.
• Potatoes have been studied using a portion of the
  E. coli enterotoxin in mice and humans.
• Other candidates for edible vaccines include
  banana and tomato, and alfalfa, corn, and wheat
  are possible candidates for use in livestock.
• Edible vaccines may lead to the eradication of
  diseases such as hepatitis B and polio.

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So... How Do Edible Vaccines Work?
For the last decade, scientists have known how to
genetically engineer a plant to produce a
desired protein. The two most common tools used
to do this are
Agrobacteria
Gene Gun
Cut out the selected region of the plasmid.
Infect the plant with the agrobacteria and grow
it in a medium.
DNA is coated on microscopically tiny gold beads
that are placed in a vacuum chamber. The gene
gun then allows compressed gas to expand, pushing
the beads down until they hit a filter. The DNA
then flies off of the beads down into the tissue,
where some will enter a nucleus and become
incorporated.
Agrobacteria have a circular form of DNA called
plasmids. The plasmids are easily manipulated
because they naturally have two cut points
where a gene can be taken out and replaced with
one of the scientists choice.
Grow the plant like a regular crop.
Add the desired gene.
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Growing plants is much cheaper than producing
vaccines.
The plants that produce the edible vaccines could
be grown in third world countries.
Advantages
Plants are already regularly used in
pharmaceuticals, so there are established
purification protocols.
Agricultural products can be transported around
the world relatively cheaply.
Plants cant host most human pathogens, so the
vaccines wont pose dangers to humans.
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If the vaccines were grown in fields or on trees,
security would become a big issue.
Plants are living organisms that change, so the
continuity of the vaccine production might not be
guaranteed.
Disadvantages
The edible vaccines could be mistaken for regular
fruits and consumed in larger amounts than might
be safe.
Glycosylation patterns in plants differ from
those in humans and could affect the
functionality of the vaccines.
The dosage of the vaccines would be variable.
For example, different sized bananas would
contain different amounts of vaccine.
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Why HEK.EBNA Cells? The Principle
EBNA-1/ori-P based expression in Human Embryonic
Kidney (293) cells (293 stably transformed with
EBNA-1 gene)
The cell line is available from ATCC and, until
recently, also from Invitrogen
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Why HEK.EBNA Cells? Advantages

• In comparison to other eukaryotic expression
  systemsthe HEK.EBNA Expression System is
  rapidfrom gene to protein in 4-6 weeks

• The cells can be grown adherently and in
  serum-free suspension culture

• It can be applied to generate stable cell lines
  (pools/ clones) and in transient mode on small
  and large scale

• In transient mode not only secreted and
  membrane-bound, but also intracellular proteins
  can successfullybe expressed

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HEK.EBNA Expression Vectors

• Basic vector (alsoGateway adapted)
• Can be decorated withN- or C-terminal tags,
  heterologous leadersequences
• Co-expression of e.g. GFP via IRES element
• Selectable marker for generation of stable cell
  line

Commercially available HEK.EBNA vectors pREP4
and pCEP4 (Invitrogen)
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A Transient Transfection Run..
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.in Multiparallel Fashion
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Cell/Supernatant Harvest and Cell Lysis