Preparation and Application of Proteomic Antibody Bank against ...

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Bank against Whole Water-Soluble Proteins from. Rapid-Growing Bamboo Shoots. Yu-Jen Wu ... (3) Some proteins are less abundant. Proteome → Ab bank.
Preparation and Application of Proteomic Antibody Bank against Whole Water-Soluble Proteins from Rapid-Growing Bamboo Shoots

Yu-Jen Wu, Dai-Jer Wu, Jiann-Shing Wu and Rong-Huay Juang*

* Major affiliation:

Department of Biochemical Science and Technology (BST), National Taiwan University, Taipei, Taiwan 106 1

The power of proteomic tools could be enhanced Proteolytic digestion

Sample

2-D electrophoresis

Pure protein

(Identify significant difference)

MALDI-TOF Mass spectrum

Proteolytic fragments Capillary Electrophoresis HPLC

GCG

LC/ESI/MS Database searching

Peptide sequence N2

WIKIPEDIA

竹文化 Oriental bamboo culture

WIKIPEDIA

3

Protein pattern changes during rapid-growth period Underground (0)

Green bamboo

10 cm

40 cm

60 cm

Cellulose biosynthesis

Unpublished (2007)

Protein ID

Accession no.

Calculated Mr (kD) / pI

Sequence coverage (%)

(MASCOT)

79

Sucrose synthase

AAV64256 (Bambusa oldhamii)

92.8 / 6.03

35

402

14

80

Sucrose synthase

AAV64256 (Bambusa oldhamii)

92.8 / 6.03

35

245

7

82

Sucrose synthase

AAV64256 (Bambusa oldhamii)

92.8 / 6.03

35

1112

45

8

UDP-glucosepyrophosphorylase

BAB69069 (Oryza sativa)

51.6 / 5.4

18

302

26

9

UDP-glucosepyrophosphorylase

BAB69069 (Oryza sativa)

51.6 / 5.4

17

359

20

10

UDP-glucosepyrophosphorylase

BAB69069 (Oryza sativa)

51.6 / 5.4

21

408

38

11

UDP-glucosepyrophosphorylase

BAB69069 (Oryza sativa)

51.6 / 5.4

20

377

35

Bambusa oldhamii

(0)

20 cm

Score

Match

4

Compile 2-DE results into metabolic pathway flux Unpublished (2007)

Sucrose synthase

Starch

Activity staining 0 10 20 40 60 cm

Starch phosphorylases

Activity staining 0 10 20 40 60 cm

0

▲ H-SP ▼ L-SP

10 cm

Immunostaining (L-SP) 20 cm

Glc-1-P

Starch content 0

60 cm

0

40 cm

Immunostaining

▲ UDPGase

0 cm

UDPGase (UDPG pyrophosphorylase)

60 cm 0

20

60 cm

Specific activity 20 cm

UDPG UDPG

Invertase Activity staining 0 10 20 40 60 cm

60 cm

60 cm

▲ Sucrose synthase (SuSy)

Glucose Fructose



0

UDP-glucuronate

=Invertase

Sucrose

Cell wall biosynthesis



60 cm

UDP-xylose Xyloglucan 5

Specific probe for every single protein? Bamboo shoots Underground

Full-grown (60 cm)

Silver staining

Three possible approaches: (1) Monoclonal antibody (2) Phage display (phage antibody) (3) DNA or RNA aptamer

Challenges for hybridoma technique: Western

(1) Redundant and time-consuming (2) Poor immunogenic proteins (3) Some proteins are less abundant

Starch phosphorylase (mAb detection)

Pure Ag → Single mAb

Proteome → Ab bank 6

Production of hybridoma by cell fusion Antigen

31 2 4 BA

Immunization

1 2

LB / c

3

m

4

m

B cell 1

2

3

m m

+ Myeloma

1

2 3 4

4

Spleen cells

Cell fusion Hybridoma

31 2 4

Antiserum

x

1

2

3

4

Monoclonal antibodies Pure single Ab

A mixture of all Ab Adapted from Milstein (1980) Scientific American, Oct. p.58

7

Screening and identification procedures

Proteomics (2006) 6: 5898-5902 Cell Fusion

D0

Master plates (3 plates)

D14 First screening 1-DE immunostaining

D21

D28

T-25 culture

Positive clone First identification 2-DE immunostaining

Subcloning (1 plate per clone)

D42

Second screening 1-DE immunostaining

Positive clone

D49

Figure 1 b

T-25 culture

Second identification 2-DE immunostaining

8

Stage-wise immunization and cell fusion protocol Proteomics (2006) 6: 5898-5902

(bamboo shoot)

Affinity column (I)

Affinity column (II)

Total Proteins

Partial proteins

Partial proteins

(antigen)

(not adsorbed)

(not adsorbed)

First-stage Immunization

Second-stage Immunization

Third-stage Immunization

Fusion & screening

Fusion & screening

Fusion & screening

mAb bank (I)

mAb bank (II)

mAb bank (III)

First stage

Second stage

(Third stage)

Starting material

BA

LB / c BA

Figure 1 a

LB / c

BA

LB / c

9

Total protein (antigen)

Pre-immune serum

4-week serum

A

B

C

D

E

F

6-week serum Supplementary Figure 1

8-week serum

Proteomics (2006) 6: 5898-5902 (supplement)

The immune response for complex antigens

12-week serum 10

Summary for the bank production Proteomics (2006) 6: 5898-5902

(A) First-stage Spleen cell fused with Number of clones

NS0/1 (Mouse A)

Sp2/0 (Mouse B)

screened

positive

screened

positive

First screening

150

100

180

120

Second screening (after subcloning)

320

150

500

250

78

Final monoclonal

82

(B) Second-stage Sp2/0 (Mouse C)

Spleen cell fused with Number of clones

screened

positive

First screening

100

40

Second screening (after subcloning)

400

120

Final monoclonal

32

11

The library of total 192 blots in the mAb bank Proteomics (2006) 6: 5898-5902

In total 1,360 spots 12

Total proteins

A

B

Proteins after absorption

(antigen for First-stage mAb bank preparation)

(antigen for Second-stage mAb bank preparation)

First-stage immunization

Cell fusion and screening

Mouse A

D

C

Second-stage immunization

Mouse C

E

F

Proteomics (2006) 6: 5898-5902

The immune response & the mAb production

* * *

Mouse B

G

First-stage bank

Second-stage bank

(mixture of 160 mAb)

(mixture of 32 mAb)

Antiserum

*

H *

Antiserum Total mAb bank

Figure 2

(mixture of 192 clones)

13

An example for the application mAb H7-E3

Fructose-6-phosphate

~ GAPN Glyceraldehyde-3-phosphate Phosphorylating NAD+ & Pi glyceraldehyde-3-phosphate 11 NADH dehydrogenase (GAPDH) Glyceraldehyde-1,3-bisphosphate 3-Phosphoglycerate kinase

2

ADP

NADP+

33 NADPH

ATP

3-Phsophoglycerate

33

Pyruvate

GAPN: Nonphosphorylating Glyceraldehyde-3-phosphate dehydrogenase 14

3

10

3

10

3

10

GAPN-Hi

mAb

H7-E3

0

12.5% SDS-PAGE (2nd dimension)

Glyceraldehyde-3-P dehydrongenase (GAPN)

pI

20 cm

40 cm

Bamboo shoot (0) Bamboo leaf 60 cm GAPN-Lo bamboo shoots

ECL staining

DAB staining

DAB staining

Unpublished (2007) 15

Purified GAPN-Hi was transform into GAPN-Lo 3

10

pI

+ Crude extract (0) + Crude extract (60 cm)

*

*

+ Boiled extract

12.5% SDS-PAGE (2nd dimension)

+ Fresh extract

Purified GAPN-Hi (incubation: 4ºC, 12 h) * GAPN-Lo

Unpublished (2007) 16

Proof of the concept “The proteomic mAb bank” Conclusions: (1) We have successfully prepared 192 hybridoma cell lines which produce specific mAb against complex proteins in a proteome (2) The screening and identification procedures were critical in selecting useful mAb lines (3) Stage-wise immunization and cell fusion worked successfully for the poor antigens (4) This work was accomplished by two full-time research staffs in 18 months (5) The number of antibodies in the final bank could be increased if more resource is invested and automation is implemented 17

Possible limitations (1) The reliability of the technique depends on how the total protein are prepared, since proteins are heterogeneous in properties (2) 2-DE is not perfect which could not faithfully display all proteins in a gel at the same time (3) Immune system can not distinguish between proteins with high homogeneity in their sequences (4) Some antibodies might not recognize the native conformation of their antigen (5) Automation for the 2-DE and Western detection is urgently needed, however, the current progress is very limiting

18

Purification of enzyme X by DEAE Sephacel 13 cm strip, pH 3-10 12.5% SDS-PAGE First Ab 1:2,000 (H7-E3) Second Ab 1:5,000 (Goat anti-mouse IgG)

DEAE Sephacel

0.5

1.4 0.4 1.2 0.3

NaCl (M)

Protein Concentration (A570)

1.6

1.0 0.2 0.8

0.1

0.6

0.4

0.0 0

Fractions added to GAPN 

20

40

60

80

Fraction Numbers

19