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					Protein Purification Involving a
Unique Auto-Cleavage Feature of a
Repeated EAAAK Peptide
Interdisciplinary Research of
     Enzyme Technology

                       Protein
                      Production




  Structure &                         Bio-conversion
    Catalytic   Enzyme Research
  Mechanism




                Bio-medical application
        Interdisciplinary Research of
             Enzyme Technology
                                                          Expression systems
                                                          Purification Techniques
                                       Protein
                                      Production




               Structure &                            Bio-conversion
                 Catalytic      Enzyme Research
Glycoside
hydrolases GH-1,Mechanism                                                 Oligosaccharide
GH-3, GH-17, GH-18,                                                       preparation
GH-29, GH-46, GH-54,                                                      Biomass
GH-64, GH-72, GH-75                                                       degradation
                                                                          Transglycosylation
                                Bio-medical application
                  LC/MS/MS
               Biosensor/SPR
                  SiNW-FET
               Nano-particles
Purification
Animal tissue   Plant materials   Microorganisms

           Grinding                Fermentation


                         Extracellular     Intracellular
          Extraction
                           enzymes           enzymes


                  Filtration               Disruption


Concentration      Purification          Pure Enzyme
Strategy for massive production and
purification of protein
Current strategies and problems

Recombinant protein technology is the best
solution so far.
Current strategies and problems

 To simplify purification of recombinant proteins,
 several engineered affinity tags are used with
 which fusion protein can be purified to near
 homogeneity in a simple procedure.



                                  Linker




                        Carrier Protein Target protein
Protein purification based on affinity binding
                                 Linker




   Affinity matrix      Carrier Protein Target protein

                     Binding
Protein purification based on affinity binding

                   Binding              Linker




 Affinity matrix             Carrier Protein     Target protein

   • Glutathione S-transferase (Novagen, GST)
   • Maltose-binding protein (pMAL system, NEB)
   • Chitin-binding domain (IMPACT system, NEB)
Protein purification based on affinity binding




         excess       wash
Dialysis to remove
       Current strategies

Carrier protein (or Tag) may need to be removed,
commonly by protease, after fusion protein has
been purified before subsequent use in downstream
application.
       Common Drawbacks

                                    Linker




Affinity matrix

        • Costly affinity matrix required.


        • Post proteolytic process needed.
Protein purification based on affinity binding

                      Binding              Linker




    Affinity matrix             Carrier Protein     Target protein

   • Glutathione S-transferase (Novagen, GST)
   • Maltose-binding protein (pMAL system, NEB)
    • Chitin-binding domain (IMPACT system, NEB)
    • Chitin-binding protein with auto-cleavage peptide linker
      (developed by NCTU)
 A new system developed by our group


A vector containing chitin-binding protein and
repeated EAAAK peptide linker to form a
simple and cost-effective system for protein
expression and purification.



          CBP              Linker               MCS




                  Repeated EAAAK peptide
                  with auto-cleavage property
History of our finding……
Starting from the study of Chitinase from
Bacillus NCTU2

  Characteristics of chitin-binding Protein (CBP)

  • CBP promotes the hydrolysis of chitin catalyzed
    by chitinase.
  • CBP has good binding specificity for chitin.
  • pH>8, CBP can bind to chitin.
  • pH<6, CBP can be eluted.
     
          




                    
                         


                         
                               
                                     

                                    



                                         
                                                                Structures of Chitinases
                                                        
                                             
                                                       



                                                                     TIM-barrel structure


                                              
                               
                    




                                                                                                 Serratia ChiA




Bacillus
NCTU2
ChiA                                                                   Linker




                Catalytic
                active ?                                                               Chitin-binding domain
                                                                                             (1~150 aa)
           Vector design
Linker         amino acid sequence
  1            AKRGWI
  2            GTGGEGPGG
  3            GTGGEGPGGGGPGEGGTG
  4            TTLGSNLGTLGLK
  5            (EAAAK)5
  6            GTGGEGPGGGGPGEGGTGGTGGEGPGG
  7            (GGGGS)5

                                                            Nde1


         PT7   CBP21 gene   Linker 1~7   protease cutting site




                                                   A
Procedure of pRSET/CBP-V5G vector construction




      CBP      Linker   Protease cutting site   MCS
                         The chimeric chitinase is active
                         without significant improvement in
                         catalysis.
                             However, interestingly……




                    Linker




The fusion protein broke into
two fragments at pH 6.0!
The fusion protein broke into two fragments at pH 6.0!




                            CBP-V5G-ChiA (MW 58 kDa)

                            NCTU2 ChiA (36 kDa)



                            CBP (19 kDa)
                             Lane 1:Sample kept in water for hours (pH 6.9).
                             Lane 2:Sample in Pi buffer (50 mM, pH 6.0) for 1 day
                             Lane 3:Sample in Pi buffer (50, mM, pH 6.0) for 2 days
                             Lane 4:Sample kept in water (pH 6.9) for 1 day
                             Lane 5:Sample kept in water (pH 6.9) for 2 day
Other cases



                                              CBP-V5G-CNS( 45 kDa)



                                              CNS (chitosanase, 24kDa)
      22.5
                                              CBP (19 kDa)




             Exchange buffers with pH 4.2 -
             8.0 and kept at 25 ℃ for 12 h.
Dose Auto-cleavage occur on
  CBP-(EAAAK)5-G-ChiA?

Or contamination of protease?
        M   pH8.0   7.5   7.0   6.0   5.0   4.2


66.2

45
                                                  CBP-V5G-CNS ( 45kDa)
35

22.5                                              100 ℃ for 10 min under pH 3.6;
                                                  exchange buffers with pH 4.2 -
                                                  8.0 and then kept at 25 ℃ for
18.4
                                                  12 h.

14




     pH-dependent auto-cleavage of (EAAAK)5 linker!!
   Construction of fusion CNS with various
   repeated EAAAK linkers

• CBP- (EAAAK)2 G-CNS
• CBP- (EAAAK)3 G-CNS
• CBP- (EAAAK)4 G-CNS
• CBP- (EAAAK)5 G-CNS
• CBP- (EAAAK)5 -CNS (Fusion protein without genenase I
                        cutting site)
• (EAAAK)5 G-CNS (Fusion protein without CBP)
The fusion proteins were incubated in
phosphate buffer (pH 6.0 at 16 ℃) so that partial
auto-cleavage fragments can be obtained.
SDS PAGE and MS analyses of auto-cleavage of
             the fusion Protein
                                    Lane 1: protein marker
                                    Lane 2: CBP-V2G-CNS
                                    Lane 3: CBP-V3G-CNS
                                    Lane 4: CBP-V4G-CNS
                                    Lane 5: CBP-V5G-CNS
                                    Lane 6: CBP-V5-CNS
                                    Lane 7: V5G-CNS
SDS PAGE and MS analyses of auto-
  cleavage of the fusion Protein
                              Lane 1: protein marker
                              Lane 2: CBP-V2G-CNS
                              Lane 3: CBP-V3G-CNS
                              Lane 4: CBP-V4G-CNS
                              Lane 5: CBP-V5G-CNS
                              Lane 6: CBP-V5-CNS
                              Lane 7: V5G-CNS
SDS PAGE and MS analyses of auto-
  cleavage of the fusion Protein
                              Lane 1: protein marker
                              Lane 2: CBP-V2G-CNS
                              Lane 3: CBP-V3G-CNS
                              Lane 4: CBP-V4G-CNS
                              Lane 5: CBP-V5G-CNS
                              Lane 6: CBP-V5-CNS
                              Lane 7: V5G-CNS
SDS PAGE and MS analyses of auto-
  cleavage of the fusion Protein
                              Lane 1: protein marker
                              Lane 2: CBP-V2G-CNS
                              Lane 3: CBP-V3G-CNS
                              Lane 4: CBP-V4G-CNS
                              Lane 5: CBP-V5G-CNS
                              Lane 6: CBP-V5-CNS
                              Lane 7: V5G-CNS
SDS PAGE and MS analyses of auto-
  cleavage of the fusion Protein
                              Lane 1: protein marker
                              Lane 2: CBP-V2G-CNS
                              Lane 3: CBP-V3G-CNS
                              Lane 4: CBP-V4G-CNS
                              Lane 5: CBP-V5G-CNS
                              Lane 6: CBP-V5-CNS
                              Lane 7: V5G-CNS
SDS PAGE and MS analyses of auto-
  cleavage of the fusion Protein
                              Lane 1: protein marker
                              Lane 2: CBP-V2G-CNS
                              Lane 3: CBP-V3G-CNS
                              Lane 4: CBP-V4G-CNS
                              Lane 5: CBP-V5G-CNS
                              Lane 6: CBP-V5-CNS
                              Lane 7: V5G-CNS
Protocol of
 one-pot
  protein
purification
  CBP-V5G-CNS                           CBP-V5G-LPHase




Lane 1: marker
Lane 2: crude enzyme
Lane 3: β-chitin purified enzyme
Lane 4: After auto-cleavage, the obtained target protein
        CNS: 24 kDa, LPHase: 40 kDa
Purification of His-Tagged Recombinant protein using
Nickel column
                       His-Tagged protein can bind to nickel
                       column with moderate affinity and can be
                       eluted with high concentration of
                       imidazole.
His-Tag + auto-cleavage peptide +
magnetic particles

         Will it work??
One-step protein purification using MP and ACP
                                               Target protein

                                               Non-tagged protein



    12000 rpm            ultra-sonication

                           12000 rpm



                                            crude protein

                                                                    His-tag binding




                        Wash & cleavage


            remove MP
His8-GFP-(EAAAK)2-mcherry
       1   2   3   4   Lane 1: marker
                       Lane 2: crude enzyme
75kD
                       Lane 3: bound protein
63kD
                       Lane 4: unbound protein after auto-
48kD                   cleavage
35kD


28kD



17kD

10kD
    His6-(EAAAK)3-GFP
        1   2   3   4
100kD
 75kD
 63kD
48kD
35kD


28kD
                        Lane 1: marker
                        Lane 2: crude enzyme
                        Lane 3: MP bound with protein
17kD                    Lane 4: unbound protein after auto-
                        cleavage
10kD
   Conclusions
• The repeated EAAAK peptide exhibited an auto-cleavage
 feature which can be mediated by pH condition.

• With this system, many proteins have been successfully
 purified.

• Integration of auto-cleavage peptide (ACP) technique with
 NTA-coated magnetic particles coated can simplify the
 purification process.

				
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posted:11/8/2012
language:English
pages:41