Characterization of a twocomponent immobilization system through GST activity

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Characterization of a two-component
immobilization system through GST activity

• Burcu Kaplan, Erinc
  Sahin,
• Alpay Taralp and
  Zehra Sayers
• 
  Department ofChemistry/Biochemistry,Delaware,
• 
  Biotechnology Institute, University of Delaware

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Immobilization

• Immobilization of enzymes is often used in
  biotechnology in
• Mediation of synthetic transformations in
  industry
• Diagnostic purposes
• Advantages of immobilization
• prevention of enzyme loss in continuous systems,
• increased thermal or pH stability,
• increased longevity,
• under certain circumstances higher activity

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Current techniques

• Adsorption technology
• Encapsulation technology
• Cross-linking technology
• Covalent bonding
• Site specific immobilization using antibodies

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Immobilization System

• The system consists of two components
• pETM-GFP Imm construct
• Carboxylated polymeric surface

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pETM-GFP-Imm Protein/Enzyme Construct

• pETM-11 expression vector
• Affinity tag
• Fluorescent peptide
• Multiple Cloning Site (MCS)
• Cleavable linker
• Flexible joint
• Enzyme

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Affinity Tag

• Multiple histidine residues in series

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Fluorescent Protein

• Green fluorescent protein (GFP) aids in
  visualization and quantification of the
  immobilized enzyme

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Multi-Cloning Site (MCS)

• Provides insertion sites for recombinant proteins
  downstream of GFP
• List enzymesSacI,SalI,HindIII,NotI,EagI,

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Cleavable linker
Multi-Cloning Site (MCS)

• Provides insertion sites for recombinant proteins
  downstream of GFP
• Enzymes
• SacI,SalI,HindIII,
• NotI,EagI,

• TEV (Tobacco Etch Virus) protease
• recognition site permits easy release of
  immobilized peptide from the surface into the
  solution phase following proteolytic cleavage

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Cleavable linker

• TEV (Tobacco Etch Virus) protease
• recognition site permits easy release of
  immobilized peptide from the surface into the
  solution phase following proteolytic cleavage.

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Flexible joint

• Helps to insert recombinant protein/enzyme in
  frame with His-tag.
• Provides increased mobility and separation from
  the surface through linkage via GFP and the
  flexible Gly-Gly-Thr sequence.
• 5 GTACG CCATGG GAGGCAC GGTACC TTGTG 3(29 bp)
• 3 CATGC GGTACC CTCCGTG CCATGG AACAC 5

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(No Transcript)
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Map of pETM-GFP-Imm.
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Enzyme/Protein

• Glutathione S-transferase (GST) used as test
  enzyme.
• GST catalyzes the conjugation of wide variety of
  electrophilic substrates with glutathionine
• Gene 717 bp (from Schistosoma japonicum)
• Protein 26 kDa

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Map of pETM-GFP-Imm-GST
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Carboxylated polymeric surface

• Polymeric surface treated by ammonium
  persulfate(APS), results in the formation of
  carboxyl groups.

FT-IR spectrum of unmodified, 2M and 3M APS
modified polystyrenesamples. Black, blue and red
lines represent unmodified, 2M and 3M APS
modified polystyrene samples respectively.
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FT-IR spectrum of unmodified, 2M and 3M APS
modified polystyrenesamples. Black, blue and red
lines represent unmodified, 2M and 3M APS
modified polystyrene samples respectively.
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Immobilization

• Achieved through a direct interaction between the
  surface-pendent carboxyl groups and the imidazole
  moieties of poly(6)histidine tag
• Fluorescent measurements at 398 nm verify surface
  binding.

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Practical Methods

• Insertion of GST into pETM-GFP Imm
• Expression of recombinant fusion protein
• GST activity assay from cell extracts
• Purification of recombinant fusion protein
• Surface binding
• GST activity assay of immobilized recombinant
  fusion protein

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PCR amplification

• PCR amplification of GST gene
• Primers 
• Forward 5CCC GAGCTC ATG TCC CCT ATA CTA
  3
• Reverse 5AAA AAGCTT TCA CGA TGC GGC
  CGC 3
• Template pGEX- 4T-2(Amersham Pharmacia)

700 bp
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Preparation of pETM-GFP-Imm and GST fragment for
the final construct

• Digestion with HindIII
• and SacI

• Ligation
• Subcloning into E.coli XL1Blue cells

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Verification of the construct

• Digestion of isolated plasmids (SacI/HindIII)
• 6000 bp
• 4000 bp
• 800 bp
• 700 bp
• 2. Sequencing (SEQLAB (Germany)

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Expression of the Recombinant Fusion Protein

• Expression cells BL21(DE3)

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Expression
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Results

• His-Tag-GFP-GST fusion protein is expressed in
  E.coli BL21DE3 cells
• The UV visualization of expression reveals that
  GFP is still fluorescent in the presence of a 26
  kDa fusion partner.

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Expression and Purification of the His-Tag-GFP
Fusion
Pellets of bacterial colonies picked from plates
of control and induced BL21(DE3).
The fusion protein His-tag-GFP was purified using
the AKTA FPLC system (Amersham Biosciences) with
MonoQ Ion Exchange Chromatography Column (10/10)
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Binding of the His-Tag GFP fusion protein to the
Modified Polystyrene surface

• Dialysis requantification of purified
  recombinant fusion protein
• Oxidation of polystyrene 96-well plates using 2M
  and 3M ammonium persulfate solutions.
• Binding at the pH of the dialysis solutions.
• Detection and quantification of binding by
  fluorescence measurements, at the characteristic
  wavelength of GFP,398 nm.

UV transmission photographs of bound his-tag-GFP
fusion with and without buffer in the wells.
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Fluorescence measurements
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The need for a test enzyme

• Presence of His-Tag as fusion partner affects
  fluorescence transmission.
• GFP fluorescence measurement data are not
  suitable for quantification of the system due to
  absence of a reference molecule.
• GST can be quantified by means of enzymatic
  activity and compared to free GST fusion.

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Testing Enzyme Activity
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Next...

• Quantification of surface binding by GST activity
  meaurement
• From cell extract
• Purified fusion protein
• Glutathione S-Transferase Assay Kit (Cayman
  Chemical Company,Ann Arbor,USA) will be used for
  activity measurements

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GST activity assay from cell extract

• Before purification step, GST activity of
  recombinant fusion protein will be assayed in
  order to assure activity in the presence of GFP
  fusion partner.
• Kit measures total GST activity (cytosolic and
  microsomal)by measuring the conjugation of
  1-chloro-2,4-dinitrobenzene (CDNB) with reduced
  glutathione. The conjugation is accompanied by an
  increase in absorbance at 340 nm.The rate of
  increase is directly proportional to the GST
  activity in the sample.

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GST activity assay of immobilized recombinant
fusion protein

• Activity of free GFP-GST will also be assayed and
  compared to that of immobilized, comparing the
  absorbance values at 340 nm.