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The TAGZyme System
September 2004
COOHTarget ProteinPADAPaseDAPase
The TAGZyme system (I)
QQHH HH QQ HH QQMM KK HH QQ HH QQ HH QQDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPase DAPaseDAPaseDAPaseDAPase
COOHTarget ProteinIK
COOHTarget ProteinLR
Natural DAPase
stop
Natural DAPase
stop
QQQQpGAPasepGAPaseQCyclaseQCyclaseDAPaseDAPase
The TAGZyme system (II)
QQHH HH QQ HH QQMM KK HH QQ HH QQ HH QQDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPase DAPaseDAPaseDAPaseDAPase COOHNH2 Target Protein
QCyclaseQCyclaseQCyclaseQCyclase
QCyclaseQCyclaseQCyclaseQCyclase
The TAGZyme downstream process
Skip DSP
IMAC purification(immobilized metal affinity chromatography)
Cleavage with His-tagged DAPase
Removal of enzymes and contaminants by subtractive IMAC
CrudeHis-tag protein
PurifiedHis-tag protein
COOHNH2Target Protein
Purified
Production of human growth hormone (hGH)
Skip DSP
Histag-hGH
hGH
1 2 3 54 6 7
Fast buffer-exchange
TAGZyme cleavage/refolding
Subtractive IMAC
Crude urea/GuClSolubilized Histag-hGH
PurifiedHistag-hGH
IMAC purification
Fast buffer-exchange
COOHNH2hGH
Lane 2
Lane 3: 5 min
Lane 5: 20 minLane 4: 10 min
Lane 6: 30 min
Lane 7
Purification scales•Up to 200 mg
Typical recoveries•IMAC purification : 95-98 %•Cleavage/subtractive IMAC : 90-95 %
The TAGZyme system
• Used for both intracellular and secreted proteins
• Successfully tested in different production hosts (E. coli, insect cells,
etc)
• Scaled up to 2 gram scale
• Successfully tested on more than 200 different proteins
• The TAGZymes can be produced in bulk quantities
• For more than 10 years, DPPI has been used for production of a
pharmaceutical protein (~10 kg/year)
• Currently being tested at other pharmaceutical companies
QQQQHH HH QQ HH QQMM KK HH QQ HH QQ HH QQ QQCOOHNH2
Target Protein
The TAGZyme downstream process: strengths
• IMAC matrices have high protein-binding capacity and selectivity
• Robust and simple chromatographic procedures with high recovery (> 90 %)
• IMAC matrices are chemically stable to prolonged CIP procedures
• Tag sequences can be optimized for efficient expression levels
• Complete and specific removal of N-terminal His-tags, -i.e. the correct N-terminus is obtained without non-specific internal cleavage
• Simultaneous removal of both processing enzymes and residual contaminants by subtractive IMAC
• Process is scalable from R&D to production
Contact information:José Arnau, PhDUnizyme Laboratories A/SDr. Neergaardsvej 17DK-2970 HørsholmDENMARK
Email: [email protected]: http://www.unizyme.com
Tel: +45 45760154Mobile: +45 40176233Fax: +45 45761407