• Please, do not distribute outside of P4EU, since there are unpublished data within the presentation

• Details about HEK293S GnTI- high density transfection can be found in Bláha et al., Protein Expr. Purif. 109, 7-13, 2015; pdf file is attached

• For more details, ask me at ondrej.vanek@natur.cuni.cz

• Collaboration on testing or optimization of new 293s expression methods is welcomed!;-)

PreludePrelude

293rd attempt to boost the yield 293rd attempt to boost the yield

and get the structureand get the structure

OndOndřřej Vanej Vaněěkk

Department of Department of BiochemistryBiochemistry, , FacultyFaculty of Scienceof Science

Charles University in Charles University in PraguePrague

• Introduction – NK cell receptors as benchmarking targets

• HEK293S GnTI- – suspension cultivation

• Cell culture medium development

• Plasmid development and high density transfection and production

• Regulation of cell cycle during production

• 293rd attempt: hNKRP1 story

• Lentivirus – Daedalus, UCOE, PiggyBac

OutlineOutline

Introduction Introduction –– natural killer cellsnatural killer cells

– Essential part of immune system– Large granular lymphocytes– Recognition of cancer and virus

infected cells– Activating x Inhibitory receptors– Cause apoptotic death of target

cell: „death kiss“ mediated by cell surface receptors

NK cell is checking a cancer cell

• human NKR-P1 (+ its

ligand LLT1): stay tuned…

HEK293 secretHEK293 secret(ed)(ed) benchmarkingbenchmarking

Human CD69, dimer of the extracellular domain @ 1.37 Å.The high resolution structure of the extracellular domain of human CD69 using novel polymer, Kolenko et al., Acta Cryst. F65, 1258-1260, 2009.

Benchmarking: disulfidic homodimer, few mg/l production ▲►

rat Clrb

α-M I

GnT1

α-M II

Golgi

Gly

can B

iosy

nth

esi

s P

ath

wa

y

293S GnT1-/-

oligomannose glycans Moderate expressionUniform sugarsEndo H cleavable

Swainsonineor Lec36

293T

hybrid glycans High expressionHybrid sugarsEndo H cleavable

Sf9

paucimannose glycans Low expressionUniform sugarsEndo H+D cleavable

293T

complex glycans High ExpressionComplex sugarsNOT cleavable

Kifunensine

293T

High expressionUniform (large) sugarsEndo H cleavable

oligomannoseglycans

GlyGlyccosylaosylationtion inin HEK293 HEK293 cell cell linlinee

Veronica T. Chang, Solving the glycosylation problem, Structure, 2007.

α-M I

GnT1

α-M II

Golgi

Gly

can B

iosy

nth

esi

s P

ath

wa

y

293S GnT1-/-

oligomannose glycans Moderate expressionUniform sugarsEndo H cleavable

Swainsonineor Lec36

293T

hybrid glycans High expressionHybrid sugarsEndo H cleavable

Sf9

paucimannose glycans Low expressionUniform sugarsEndo H+D cleavable

293T

complex glycans High ExpressionComplex sugarsNOT cleavable

Kifunensine

293T

High expressionUniform (large) sugarsEndo H cleavable

oligomannoseglycans

GlyGlyccosylaosylationtion inin HEK293 HEK293 cell cell linlinee

Bláha et al., Expression and purification of soluble and stable ectodomain of natural killer cell receptor LLT1 through high-

density transfection of suspension adapted HEK293S GnTI- cells. Protein Expr. Purif. 109, 7-13, 2015.

HEK293 HEK293 suspensionsuspension cultureculture

Cells keeped in suspension on orbitalshaker (135 rpm / 19 mm or 180 rpm / 50 mm orbit) in Schott-Duran square glass flasks with 0.22 µm membrane lids(autoclavable) in ExCELL293 / F17 media 1:1 mixture in 1 ml – 0.4 l cultivation volumes.

CultureCulture mediummedium

• ExCELL293 (Sigma) – good for growth and production butnot for standard PEI mediated transfection; approx. 100,- €/l… 50% discount for 50 liters… powder still cheaper

• F17 (Invitrogen) – good for transfection; originally as customstock product approx. 75,- €/l … with discount for 30 l for50,- €/l … later Freestyle F17 only for 130,- €/l! …still 50% discount for 30 l; Freestyle 293 or Expi 293 more expensive

• Enough! Let‘s make our own:

• DMEM/Ham‘s F12 1:1 mixture without calcium chloride and phenol red, with 0.1% Pluronic F68, insulin, transferrin, sodium selenite and lipid concentrate, boosted with TN1 hydrolysate, pyruvate and NEA … around 10-20,- €/l

• and let us see…

„„DHAMDHAM““ mediummedium

• DME/F-12 base medium (D9785, powder, Sigma)

Amino acids Energy Hormones, vitamines Inorganic

salts

Supplements

� L-leucine � D-glucose � insuline (I) � MgSO4� Pluronic F68

surfactant

� L-lysine� sodium

pyruvate� hydrocortisone (H) � NaHCO3 � ethanolamine

� L-methionine � L-glutamine� transferrin (T) or ferric

citrate as iron source� MgCl � lipid mixture

� L-glutamine� L-ascorbic acid (C) as

antioxidative agent� Na2SeO3

� non-essential amino acid mixture (NEA)

� tryptone N1 casein hydrolysate (Y)

� trace elements mixture (P)

Substances recommended by the manufacturer or in literature

Sterile stock solutions for optimization

Cell doubling time vs. culture medium composition:

293S GnTI-

Ex/F17 22,75 ± 1,71 hours (srel = 14,4 %)Ex/N + I*, T, P*, C 20,47 ± 2,68 hours (srel = 13,1 %)

N new medium Ex EX-CELL293 F17 FreeStyle F17 I insuline (5 mg/l) I* insuline (1 mg/l) T transferrin P trace elements P* trace elements optimized C L-ascorbic acid

„„DHAMDHAM““ mediummedium

Time of cultivation (days)

Do

ub

lin

g t

ime (

ho

urs

)

Cultivation Transfection Production

1 Ex/F17 Ex Ex

2 Ex/N + I, T Ex Ex

3 Ex/N + I, T Ex Ex/N

4 Ex/N + I, T Ex N + I, T

N new medium Ex EX-CELL293 F17 FreeStyle F17 I insuline (5 mg/l) T transferrin

� Standard transfection in commercial media gives highest yields (1)

� Cultivation in Ex/N – production only slightly lower (2)

� Production in Ex/N (3) or N (4) possible (cheaper at larger volumes)

293S transfection 293S transfection && production in production in „„DHAMDHAM““

TheThe bestbest fromfrom HEK293...HEK293...

Gaurav Backliwal et al. (NAR 2008): „Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titersexceeding 1 g/l by transient transfection under serum-free conditions“ (!!!)

Transfection with Swiss ‘XLG protocol’:- cells centrifuged and resuspended to 20.106 cells/ml in FreeStyle 293- DNA added directly to the cells followed by addition of PEI, ratio 1:2- 3 hours later cells diluted with ExCell293 4.106 cells/ml- valproic acid added to 3.8 mmol/l- cells were then propagated for 10–14 days under agitation

The ratio of plasmids used under our optimal production conditions was as follows:- 37.5% pXLGHEK-RhHC, 37.5% pXLGHEK-LC- 10% pXLGHEK-p18h- 10% pXLGHEK-p21h- 5% pXLGHEK-aFGF

• pTT28 (derivative of pTT5, like pOPINs): cytomegalovirus promotor, intron A; adenovirus tripartite leader sequence (TPL) major late promoter enhancer (TM); splicing acceptor (SA); polyA; diad symmetry (DS); oriP site for episomal replication (FR)

• pXLG: the same plus woodchuck hepatitis virus: post-transcriptional regulatory element(WPRE) and optimized ITR sequences

• our combination: pTW5sec, i.e. pTT28 + WPRE segment

PlaPlassmidmidss……

pXLG

Standard transfection is not working in HEK293S GnTI- cell line, but at high cell

density in ExCELL293 yes, even without prior DNA:PEI incubation!

A) B) C)

High density tHigh density transferansfection is superior for 293Sction is superior for 293S

(A) DNA:PEI optimization

(B) Secreted protein production (measured as SEAP activity) and (C) transfection efficiency (GFP+ viable cells)

of suspension adapted HEK293T cells (yellow) and HEK293S GnTI- cells (cyan) transfected at density of 2.0×106 cells/ml and HEK293S GnTI- cells transfected at density of 20×106 cells/ml with transfection complex formed a priori (gray) or in situ (magenta) was monitored 3 days post-transfection.

Bláha et al., Expression and purification of soluble and stable ectodomain of natural killer cell receptor LLT1 through high-

density transfection of suspension adapted HEK293S GnTI- cells. Protein Expr. Purif. 109, 7-13, 2015.

RRegulaegulation oftion of cellcell cycyclecle aandnd apoptosapoptosisis

• GFP+ 51–77 % (bcl2 only 11–32 %)

RRegulaegulation oftion of cellcell cycyclecle aandnd apoptosapoptosisis

• p18, p21, p27• bcl2, bclx• optimization of ratio of

individual plasmidsagainst total DNA amount and against each other –synergies?

• anti-apoptotic genes are causing apoptosis

• CDK inhibitors work!

RRegulaegulation oftion of cellcell cycyclecle aandnd apoptosapoptosisis

High density pHigh density producroduction oftion of LLT1LLT1 in 293Sin 293S

Skálová et al., Four crystal structures of human LLT1, a ligand for human NKR-P1, in varied glycosylation and oligomerization state. Acta Cryst D71, 578-591, 2015.

A280

V, ml

• high-density production

• p27+aFGF+VPA= 150-200% more!!!

C75

C86

C103

C163

C184

C176

High density pHigh density producroduction oftion of LLT1LLT1 in 293Sin 293S

Skálová et al., Four crystal structures of human LLT1, a ligand for human NKR-P1, in varied glycosylation and oligomerization state. Acta Cryst D71, 578-591, 2015.

A280

V, ml

• high-density production

• p27+aFGF+VPA= 150-200% more!!!

451

µm

118 µm

Final optimized production protocol:

- 20·106 cell/ml transfection in ExCELL293

- 2 µg DNA per 1·106 cells

- DNA:PEI 1:2

- transfection mix:

- 10% pTW5-p27

- 2% pTW5-aFGF

- 88% pTW5sec_LLT1

- 3-4 hour later 2·106 cell/ml production

- valproic acid added to 2 mM conc.

293 attempts to p293 attempts to produce hroduce human uman NKRNKR--P1P1

• receptor of human NK & NK-T lymphocytes, LLT1 binding partner

• expression in E. coli – inclusion bodies – refolding failed…�

• expression in HEK293T – only ca 0,1 mg/l yield and fromHEK293S GnTI- line even less… � �

• Instruct @ OPPF – library of 46 construts (various construct length, different tags) – expression test in E. coli failed � � �

• later @ OPPF library expression test in Sf9 & baculoviruses –tiny expression, not scalable – failed too… � � � � �

• in house library expression test in HEK293T line – there is expression, but still small, the original construct was the best –not better than at the beginning… � � � �

• So what now? We have the right construct and expression system, only the yield is too low…???

• Instruct @ OPPF – transfer of library into pOPINTTGneo plasmid• best construct chosen; 0,1 mg/ml G418: stable polyclonal cell line secreting

hNKR-P1 at ca 1-2 mg/l!!!☺☺☺

polyA site 1846..1956

Splice acceptor site 1251..1280

Adenoviral ML enhancer 1056..1167

Tripartite leader 756..980

CMV promoter 129..756

Ampicillin 4345..5191

Neo 6797..7591

pOPINTTGneo8008 bp

SV40 NeoR cassette 6392..7904

oriP replicon 2358..4066

The solution The solution –– 293S stabl293S stably!y!

Little Dipper star

constellation ☺☺☺☺

Selected

as one of

the four

Instruct

„success

stories“

for its

biennial

meeting!

• for polyclonal stable cell line generation with potentially dramatically higher yields

• HEK293 line commonly used as packaging cell line for recombinant viruses or virus-like particles

• lentiviruses are retroviruses similar to HIV, but pseudotyped with VSV-G (vesicular stomatitis virus) envelope protein and devoid of unnecessary HIV genetic elements

• virus generation is only transient and viral replication not possible – „safe“ gene delivery vehicle

What next? Lentiviral expression systemsWhat next? Lentiviral expression systems

LentivirLentiviralal expresexpressionsion systsystemem DaedalusDaedalus

• silencing of the inserted GOI is major drawback – various genetic elements might be used to prevent this:

• MAR – matrix attachment regions

• UCOE – ubiquitous chromatin opening element (around 2.0 kbp in vectors)

• Daedalus – shorter 0.7 kbp UCOE fragment, strong and stable expression, IRES-GFP monitoring or selection of best clones by FACS

• in our hands the cloning is totally weird…(S)LIC, Gibson assembly, RF cloning, TPCR… nothing works;-(

• to be continued…☺

Bandaranayake et al., Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active

recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Research 39, e:143, 2011.

ConclusionConclusion

• Use 293S GnTI- cell line for structural biology!

• Suspension culture with glass flasks savesconsumables

• Culture medium – ExCELL293 powder

• High density transfection superior for 293S

• High density production possible with regulation of cell cycle

• When transient is not enough – go stable!

• PiggyBac or Lentiviruses – we will see…

• Happy benchmarking!;-)

ThanksgivingThanksgiving……

• students: Jan Bláha, Edita Poláchová, Romana Čuperková, Ondřej Skořepa, and many more…

• Charles University in Prague(UNCE 204025/2012, SVV 260079/2014)

• Czech Science Foundation (15-15181S)

• High Education Development Fund (FRVS 669/2013)

• SPINE2:Complexes, P-CUBE, Instruct, BioStruct-X

• …and you for your attention!;-)

• ?2: ondrej.vanek@natur.cuni.cz