Preparation and Application of Proteomic Antibody Bank against Whole Water-Soluble Proteins from Rapid-Growing Bamboo Shoots

Transcription

1 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

2 The power of proteomic tools could be enhanced Proteolytic digestion Sample 2-D electrophoresis (Identify significant difference) Mass spectrum Pure protein MALDI-TOF Proteolytic fragments Capillary Electrophoresis HPLC GCG LC/ESI/MS Database searching N- Peptide sequence 2

3 WIKIPEDIA 竹文化 Oriental bamboo culture WIKIPEDIA 3

4 Protein pattern changes during rapid-growth period Underground (0) 10 cm 20 cm 40 cm 60 cm Green bamboo Bambusa oldhamii Cellulose biosynthesis Protein ID Accession no. Calculated Mr (kd) / pi Sequence coverage (%) Unpublished (2007) Score (MASCOT) Match 79 Sucrose synthase AAV64256 (Bambusa oldhamii) 92.8 / Sucrose synthase AAV64256 (Bambusa oldhamii) 92.8 / Sucrose synthase AAV64256 (Bambusa oldhamii) 92.8 / UDP-glucosepyrophosphorylase BAB69069 (Oryza sativa) 51.6 / (0) 9 UDP-glucosepyrophosphorylase BAB69069 (Oryza sativa) 51.6 / UDP-glucosepyrophosphorylase BAB69069 (Oryza sativa) 51.6 / UDP-glucosepyrophosphorylase BAB69069 (Oryza sativa) 51.6 /

5 Compile 2-DE results into metabolic pathway flux Sucrose synthase Activity staining cm 0 10 cm Starch H-SP L-SP Activity staining cm Unpublished (2007) Starch phosphorylases Immunostaining (L-SP) Immunostaining cm Invertase Activity staining cm 20 cm 40 cm 60 cm Sucrose synthase (SuSy) Glc-1-P UDPG UDPG UDPGase Starch content 0 60 cm 0 cm 20 cm 60 cm 0 60 cm UDPGase (UDPG pyrophosphorylase) Specific activity 0 60 cm Glucose Fructose =Invertase Sucrose UDP-glucuronate Cell wall biosynthesis UDP-xylose Xyloglucan 5

6 Specific probe for every single protein? Western Silver staining Bamboo shoots Underground Full-grown (60 cm) Three possible approaches: (1) Monoclonal antibody (2) Phage display (phage antibody) (3) DNA or RNA aptamer Challenges for hybridoma technique: (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

7 Production of hybridoma by cell fusion Antigen Immunization BA LB/c x B cell m m m m Myeloma Hybridoma Spleen cells Cell fusion Antiserum A mixture of all Ab Monoclonal antibodies Pure single Ab Adapted from Milstein (1980) Scientific American, Oct. p.58 7

8 Proteomics (2006) 6: Screening and identification procedures Figure 1 b D0 D14 D21 D28 D42 D49 First screening 1-DE immunostaining Subcloning Second screening 1-DE immunostaining Master plates (3 plates) T-25 culture (1 plate per clone) T-25 culture Cell Fusion Positive clone First identification 2-DE immunostaining Positive clone Second identification 2-DE immunostaining 8

9 Stage-wise immunization and cell fusion protocol Proteomics (2006) 6: Starting material (bamboo shoot) Affinity column (I) Affinity column (II) Total Proteins (antigen) Partial proteins (not adsorbed) Partial proteins (not adsorbed) BALB/c BALB/c First-stage Immunization Second-stage Immunization BALB/c Third-stage Immunization Fusion & screening Fusion & screening Fusion & screening mab bank (I) mab bank (II) mab bank (III) First stage Second stage (Third stage) Figure 1 a 9

10 The immune response for complex antigens Total protein (antigen) Pre-immune serum 4-week serum A B C D E F Proteomics (2006) 6: (supplement) 6-week serum 8-week serum 12-week serum Supplementary Figure 1 10

11 (A) First-stage Summary for the bank production Proteomics (2006) 6: Spleen cell fused with NS0/1 (Mouse A) Sp2/0 (Mouse B) Number of clones screened positive screened positive First screening Second screening (after subcloning) Final monoclonal (B) Second-stage Spleen cell fused with Sp2/0 (Mouse C) Number of clones screened positive First screening Second screening (after subcloning) Final monoclonal 32 11

12 The library of total 192 blots in the mab bank Proteomics (2006) 6: In total 1,360 spots 12

13 The immune response & the mab production Total proteins (antigen for First-stage mab bank preparation) Mouse A C First-stage immunization A D B Cell fusion and screening E Proteins after absorption (antigen for Second-stage mab bank preparation) Second-stage immunization Mouse C F * Proteomics (2006) 6: * * Mouse B G * First-stage bank (mixture of 160 mab) Second-stage bank (mixture of 32 mab) Antiserum * H Figure 2 Antiserum Total mab bank (mixture of 192 clones) 13

14 mab H7-E3 ~GAPN Phosphorylating glyceraldehyde-3-phosphate dehydrogenase An example for the application Fructose-6-phosphate Glyceraldehyde-3-phosphate (GAPDH) Glyceraldehyde-1,3-bisphosphate 33 3-Phosphoglycerate kinase 11 2 NAD + & P i NADH ADP ATP 3-Phsophoglycerate NADP + NADPH Pyruvate 33 GAPN: Nonphosphorylating Glyceraldehyde-3-phosphate dehydrogenase 14

15 Glyceraldehyde-3-P dehydrongenase (GAPN) pi 0 20 cm 40 cm 60 cm bamboo shoots 3 10 GAPN-Hi GAPN-Lo ECL staining 3 10 DAB staining 12.5% SDS-PAGE (2nd dimension) 3 10 DAB staining mab H7-E3 Bamboo shoot (0) Bamboo leaf Unpublished (2007) 15

16 Purified GAPN-Hi was transform into GAPN-Lo 3 10 pi + Crude extract (0) + Crude extract (60 cm) + Fresh extract + Boiled extract * * 12.5% SDS-PAGE (2nd dimension) * GAPN-Lo Purified GAPN-Hi (incubation: 4ºC, 12 h) Unpublished (2007) 16

17 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

18 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

19 Purification of enzyme X by DEAE Sephacel Protein Concentration (A 570 ) DEAE Sephacel NaCl (M) 13 cm strip, ph % SDS-PAGE First Ab 1:2,000 (H7-E3) Second Ab 1:5,000 (Goat anti-mouse IgG) Fractions added to GAPN Fraction Numbers 19