11

E. coli as a host

• PROs: Easy, flexible, high tech, fast, cheap; . . . . . . but problems

• CONs

• Folding (can misfold)

• Sorting -> can form inclusion bodies

• Purification -- endotoxins• Modification -- not done (glycosylation, phosphorylation, etc. )

• Modifications:• Glycoproteins • Acylation: acetylation, myristoylation• Methylation (arg, lys)• Phosphorylation (ser, thr, tyr)• Sulfation (tyr)• Lipid addition (prenylation: farnesyl, geranylgeranyl, palmitoylation on cys;

myristoylation on N-terminus)• Vitamin C-dependent Modifications (hydroxylation of proline and lysine)• Vitamin K-dLipid additionnependent Modifications (gamma carboxylation of glu)• Selenoproteins (seleno-cys tRNA at UGA stop)

22

Some alternative hosts

•Yeasts (Saccharomyces , Pichia)

•Insect cells with baculovirus vectors

•Mammalian cells in culture (later)

•Whole organisms (mice, goats, corn)

•In vitro (cell-free), for analysis only(good for radiolabeled proteins

33Yeast Expression Vector (example)

2 micron plasmid

2 micron seq:yeast orioriE = bacterial oriAmpr = bacterial selectionLEU2, e.g. = Leu biosynthesisfor yeast selection

Saccharomyces cerevisiae(baker’s yeast)

oriE

Your favorite

gene(Yfg)

LEU2

Ampr

GAPDterm

GAPDprom

Complementation of an auxotrophy can be used instead of drug-resistance

Auxotrophy = state of a mutant in a biosynthetic pathway resulting in a requirement for a nutrient

GAPD = the enzyme glyceraldehyde-3 phosphate dehydrogenase

4

Genomic DNA

HIS4 mutation-

Yeast - genomic integration via homologous recombination

HIS4

gfY

pt Vector DNA

FunctionalHIS4 gene

DefectiveHIS4 gene

Yfg

tp

Genomic DNA

Homologous recombination

5

Double recombination Yeast (integration in Pichia pastoris)

AOX1 gene (~ 30% of total protein)

Genomic DNA

AOX1p

Yfg

AOX1t HIS4 3’AOX1

Genomic DNA

HIS4

Yfg

AOX1p

AOX1t

3’AOX1

Vector DNA

P. pastoris-tight control-methanol induced (AOX1)-large scale production (gram quantities)

Alcohol oxidase geneHis- host

6

Protein-protein interactions

Yeast 2-hybrid systemYeast 3-hybrid and 1 hybrid systemsCo-immunoprecipitationPull-downsFar western blotsBiacore (surface plasmon resonance, SPR)Fragment complementation

7

BD =(DNA) binding domain AD =activation domain UAS =upstream activating sequence

Yeast 2-hybrid system:To discover proteins that interact with each other, orTo test for interaction based on a hypothesis for a specific protein.

?

http://www.mblab.gla.ac.uk/~maria/Y2H/Y2H.html

(bait)

(prey)

Y = e.g., a candidate protein being tested for possible interaction with X

Or: Y = e.g., a cDNA library used to discover a protein that interacts with X

?

Positive control:

8

Y = e.g., a cDNA library used to discover a protein that interacts with X

Recover the Y sequence from reporter+ colonies by PCR to idenify protein Y

No interaction between X and Y: no reporter expression

Yes, interaction between X and Y: reporter protein is expressed

9

Fusion library

Two different assays help, as there are often many false positives.

http://www.mblab.gla.ac.uk/~maria/Y2H/Y2H.html

=“prey”

Bait protein is the known target proteinfor whom partners are sought

BD= DNA binding domain; TA = transactiavting domain

10

3-HYBRID: select for proteins domains that bind a particular RNA sequence

Bait

Prey

Prey could be proteins from a cDNA library

Use a known tight protein-RNA interaction (e.g., from RNA phage MS2)

11

Yeast one-hybrid:

Insert a DNA sequence upstream of the selectable or reporter

Transform with candidate DNA-binding proteins (e.g., cDNA library)fused to an activator domain.

Each T = one copy of a DNA target sequence

12

Directed Evolution of a Glycosynthase via Chemical ComplementationHening Lin,† Haiyan Tao, and Virginia W. Cornish J. AM. CHEM. SOC. 2004, 126, 15051-15059

Turning a glycosidase into a glyco-synthase

Glycosidase: Glucose-Glucose (e.g., maltose) + H2O 2 Glucose

Indirect selection using a yeast 3-hybrid system:toward a more efficient glycosynthase enzyme

13

Indirect selection using a yeast 3-hybrid system(one of the hybrid molecules here is a small molecule)

e.g., from a mutated library of enzyme glycosynthase genes

glucose

DHFR = dihydrofolate reductase GR = glucocorticoid receptor (trancription factor )MTX = methotrexate (enzyme inhibitor of DHFR)DEX = dexamethasone, a glucocorticoid agonist, binds to GRAD = activation domain, DBD = DNA binding domain

Leu2 geneLeu2 gene

Transform a yeast leucine auxotroph. Provide synthetic chimeric substrate molecules. Select in leucine-free medium.

14

URA-3 (toxic)

Library of cellulase mutant genes(one per cell)

x x x x

cellulase

Survivors are enriched for cellulase genes that will cleave cellulose with greater efficiency (kcat / Km)

Yeast cell

Directed Evolution of Cellulases via Chemical Complementation. P. Peralta-Yahya, B. T. Carter, H. Lin, H. Tao. V.W. Cornish.

Selection of improved cellulases via the yeast 2-hybrid system

Cellobiose(disaccharide)

15

Detail

16Pathway to pyrimidine nucleotides:

URA-3 = gene for orotidine phosphate (OMP) decarboxylase

5-fluoroorotic acid

5-fluoro-OMP

5-fluoro-UMP

RNA

URA-3 decarboxylation (pyr-4)

thymidylate synthetaseinhibitiondeath

How does the URA-3 “suicide” system work?

exogenousuridine

uridine kinase

analog

17

Measuring protein-protein interactions in vitro

X=one protein Y= another protein

Pull-downs:

Binding between defined purified proteins, at least one being purified.Tag each protein differently by making the appropriate cDNA clone.

Examples:

His6-X + HA-Y; Bind to nickel ion column via X, elute (his), Western with HA Ab for Y

GST-X + HA-Y; Bind to glutathione ion column, elute (glutathione), Western with HA Ab

His6-X + 35S-Y (made in vitro); Bind Ni column, elute (his), gel + autoradiography. No antibody needed.

(HA = flu hemagglutinin) glutathione = gamma-glutamyl-cysteinyl-glycine.

18

Example of a result of a pull-down experiment

Antibody used in Western

Total protein: no antibody/Western(stained with Coomassie Blue or silver stain)

Compare pulled down fraction (eluted)with loaded. Loaded sample usually only a fraction.

Also identfy by MW (or mass spec)

19

Western blotting

To detect the antibody use a secondary antibody against the primary antibody (e.g, goat anti-rabbit IgG).

The secondary antibody is a commercial fusion protein with an enzyme activity (e.g., alkaline phosphatase).

The enzyme activity is detected by its catalysis of a reaction producing a luminescent compound.

http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html

*

*

20

Y YNon-luminescent substrate-PO4

=

Luminescent product + PO4=

Protein band on membrane

Alkaline phosphatase fusion

Secondary antibody

Antibody to protein on membrane

Detect by exposing to film

Detection of antibody binding in western blots

(chemiluminescence)

21Far western blotting to detect specific protein-protein interactions. Use a specific purified protein as a probe instead of the primary antibody

To detect the protein probe use an antibody against it.

Then a secondary antibody against the first antibody, a fusion protein with an enzyme activity.

The enzyme activity is detected by its catalysis of a reaction producing a luminescent compound.

http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html

protein protein

OR:Use a radioactively labeledprotein if interest and detect by autoradiography

22

Expression via in vitro transcription followed by in vitro translation

cDNA

T7 RNA polymerasebinding site (17-21 nt)

….ACCATGG…..

VECTOR

2. Add a translation system: rabbit reticulocyte lysate or wheat germ lysate

Or: E. coli lysate (combined transcription + translation)

All commerically available as kits

Add ATP, GTP, tRNAs, amino acids, label (35S-met), May need to add RNase (Ca++-dependent) to remove endogenous mRNA In lysate

1. Transcription to mRNA via the T7 promoter + T7 polymerase

Radioactively labeled protein

NOTE: Protein is NOT at all pure (100s of lysate proteins present), just ~“radio-pure”

23Surface plasmon resonance (SPR)Popular instrument is a Biacore

The binding events are monitored in real-time and it is not necessary to label the interacting biomolecules.

http://home.hccnet.nl/ja.marquart/BasicSPR/BasicSpr01.htm

glass plate

Reflection angle changes depending on the mass of the material on the surface.Binding increases this mass. Follow as a functrion of concentration Kd’sOr time : Measure on-time, off time; Kd = off-time/on-time

24

A Biacore result

25

Got this far

26Expression in mammalian cellsLab examples:HEK293 Human embyonic kidney (high transfection efficiency)HeLa Human cervical carcinoma (historical, low RNase)CHO Chinese hamster ovary (hardy, diploid DNA content, mutants)Cos Monkey cells with SV40 replication proteins (-> high transgene copies)3T3 Mouse or human exhibiting ~regulated (normal-like) growth+ various others, many differentiated to different degrees, e.g.:BHK Baby hamster kidey HepG2 Human hepatomaGH3 Rat pituitary cellsPC12 Mouse neuronal-like tumor cellsMCF7 Human breast cancerHT1080 Human with near diploid karyotypeIPS induced pluripotent stem cells and:Primary cells cultured with a limited lifetime. E.g., MEF = mouse embryonic fibroblasts, HDF = Human diploid fibroblasts

Common in industry:NS1 Mabs Mouse plasma cell tumor cellsVero vaccines African greem monkey cellsCHO Mabs, other therapeutic proteins Chinese hamster ovary cellsPER6 Mabs, other therapeutic proteins Human retinal cells

27

Mammalian cell expression

Generalized gene structure for mammalian expression:

cDNA geneMam.prom.

polyA site

intron

5’UTR3’UTR

Intron is optional but a good idea

28

Popular mammalian cell promoters

• SV40 LargeT Ag (Simian Virus 40)• RSV LTR (Rous sarcoma virus)• MMTV (steroid inducible) (Mouse mammary tumor virus)• HSV TK (low expression) (Herpes simplex virus)• Metallothionein (metal inducible, Cd++)• CMV early (Cytomegalovirus)• Engineered inducible / repressible:

tet, ecdysone, glucocorticoid (tet = tetracycline)

29Engineered regulated expression:Tetracycline-reponsive promotersTet-OFF (add tet shut off)

tTA cDNA

tTA = tet activator fusion protein:tetRdomain

VP16 tc’nact’n domain

No tet.Binds tet operator(if tet not also bound)

tetRdomain

VP16 tc’nact’n domain

Tetracycline (tet), or,better, doxicyclin (dox)

active

not active

CMV prom.

polyA sitetTA gene must be in cell (permanent transfection, integrated):

Tet-OFF

Tet-OFF

(Bujold et al.)

Allosteric change in conformation

30

MIN. CMV prom. your favorite gene

polyA site

Mutliple tet operator elements

MIN. CMV prom. your favorite gene

polyA site

tetRdomain

VP16 tc’nact’n domain

not activelittle transcripton (2%?, bkgd)

Doxicyclin present:

MIN. CMV prom. your favorite gene

polyA siteactivePlenty of transcripton

No doxicyclin:

tetRdomain

VP16 tc’nact’n domain

RNA po l

Tet-OFF

31

Tetracycline-reponsive promotersTet-ON (add tet turn on gene

tTA cDNA

tetRdomain

VP16 tc’nact’n domain

tetRdomain

VP16 tc’nact’n domain

Tetracycline (tet), or,better, doxicyclin (dox)

active

not active

Full CMV prom.

polyA site

Different fusion protein: Does NOT bind tet operator(if tet not bound)

Tet-ON

Must be in cell (permanent transfection, integrated): commercially available (293, CHO) or do-it-yourself

32

MIN. CMV prom. your favorite gene

polyA site

Mutliple tet operator elements

MIN. CMV prom. your favorite gene

polyA site

active

Doxicyclin absent:

MIN. CMV prom. your favorite gene

polyA siteactivePlenty of transcripton (> 50X)

Add dox:

tetRdomain

VP16 tc’nact’n domain

RNA pol II

Tet-ON

tetRdomain

VP16 tc’nact’n domain

not active little transcription (bkgd.)

doxicyclin

33

SW Michnick web site: http://michnick.bcm.umontreal.ca/research/images/pca_general_en.gif

F = reporter protein fragment

Enzyme fragmentsthemselves do not associate well enough to reconstitute an active enzyme

Reporterenzyme

34

FK506 = immunosuppressant drugFKBP = FK506 binding proteinFRAP = FKBP–rapamycin binding proteinFRB= FKBP–rapamycin binding domain of FRAP

DHFR = dihydrofolate reductaseDHF=dihydrofolate = FH2

THF=tetrahydrofolate = FH4

fMTX=fluorescent methotrexate

fMTX

DHFR fragments

Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays, I. Remy and S.W. Michnick PNAS 96, 394–5399, 1999

IN PURINE-FREE MEDIUM

Fluorescein-MTX

Rapamycin promotes the association of the 2 protein domains

35

a phosphatase

FK506 recruits FKBP to bind to calcineurin and inhibit its action as a specific phosphatase

36

Claim detection of 0.05 nM rapamycin??

No.

of

CH

O c

olon

ies

[rapamycin]

37

Background association of FKBP and FRB without rapamycin

(compare)

Leucine zipper protein fragments instead ofrapamycin binding proteins (positive contro)

CHO cells(permanent transfection)

cos cells(transient transfection)

Fluorescent methotrexate (fMTX) assay

38

8-fold increase in fluorescence per cell

Measure affinity for a drug in vivo

fluorescence-activated flow cytometer(FACS is this plus more)N

o. o

f ce

llsF

luor

esce

nce

inte

nsity

Log of fluorescence intensity

[rapamycin]

39

EMP1 = Erythropoietin mimetic peptide 1

Erythropoietin-erythropoietin receptor (dimer) interaction: Efficacy of a peptide mimetic

Erythropoietin

In vivo assay of drug effectiveness (EMP1)(inexpensive substitute for erythropoietin?)

Erytropoietin (EPO) receptor

40

FACS = Fluorescence-activated cell sorter

Impart a charge on the recognized cell

Less than one cell or particle per droplet. Thus the most that most droplets contain is one particle.

Charged plates attract droplets containing a particle of the opposite charge

Cells remain viable if treated with care.

Can be used purely anaytically without the sorting capability. Thencalled “flow cytometry”, or also called FACS anyway.

41

No.

of

cells

Having this much fluorescence

Histogram-type display

No fluorescence (background autofluorescence)

Red stained

Usually a log scale

42

One cell

Amount of red fluorescence (log)

Am

ount

of

gre

en f

luor

esce

nce

(log)

Say, want high reds butlow greens:Instruct the FACS to deflect cells in this quadrant only. Collect and grow or analyze further.

Analysis on 2 colors

Scatter plot display

You decide on the positions of of demarcations

43

A. Flow cytometry data: 2-D plots where each point represents one particle. Then contour lines plotted around the point density. Here light “forward” scattering (irrespective of wavelength) is measured (FSC). Instrument can be set to reject data from 2-bead doublets that scatter light more.

B-D. Amplified beads hybridized to 2 probes, one specific to the S allele of a certain gene and one specific to the L allele. The beads carry the amplified PCR products corresponding to this region from 3 human individuals. The blue points come from microspheres that contained both types of PCR products from both alleles, despite the high dilution.

Green signalR

ed s

ign

al

Both signals

Neither signal

Analysis of beads representing the human genome using allele-specific hybridization probes and the FACS

Beaming bead FACS analysis

44