universal purification protocol for a diverse set of proteins

1
Challenges for HTP protein purification
Quantity and Quality

• universal purification protocol for a diverse
  set of proteins
• scale equivalent to cloning and structure
  determination
• feasible and economical
• as few steps as possible
• as fast as possible
• minimize aggregation problem ..

2
Expression construct selection
His
thrombin
protein
pET15b

• Affinity tag
• small
• disorder
• easy to remove
• (protease)
• Protease
• specificity
• fast
• cheap
• tagged

His
thrombin
protein
S
Xa
pET30LIC
His
protein
Xa
MCSG3
His
protein
TEV
pRroEX(ENLYFQ?G)
His
protein
TEV
MCSG7 (ENLYFQ?S)
Tag cleavage efficiency
Dementieva et al. 2002
3
Purification protocolas few steps as possible
crude

• IMAC I usually provides a major
• step of the purification
• IMAC II removes His-TEV and
• persistent contaminant proteins
• in E.coli host
• Gel-filtration polishing
• before crystal optimization

IMAC I
tag cleavage
IMAC II
gel filtration
Milligram-scale protein production
crystallization (screen)
24
20
Crystallization (optimization)
Protein number
8
Se-Met protein
2
2
2-5
5-10
10-20
20-40
gt50
mg pure protein per 1 L culture
4
With or without tag ?
5
Affinity buffer exchange (AKTA platform,
Pharmacia)
Buffer exchange
Semi-automated - chromatography system for
simultaneous protein purification
MIXER
Affinity Buffer exchange
Fraction Collector
5
4
Outlet Valve
NO
NC
NO
NC
8
1
7
2
6
3
5
4
30 min
BufferValve
F4
F5
F6
F3
8
8
1
1
7
7
2
6
2
6
3
3
5
5
4
4
11
12
13
14
15
16
17
18
7 Affinity columns
Dementieva et al. 2002
6
Quality control as fast as possible
7
MCSG Protein Purification Database Notebook

• Protein Purification Database Notebook is fully
  searchable throughout results of purification.
• User is able to use APC number Host identifier or
  Vector description as keys for his search.
• The MCSG Report Field is connected directly to
  MCSG Report Website (http//www.mcsg.anl.gov/targe
  t) and is updating corresponding filed for
  purification progress report.
• SDS Gel Button is connected through the File
  Report mechanism to Agilent 2100 Bio Sizing
  Software for separation results.
• Chromatography Button invokes connection to File
  Report mechanism to Pharmacia Purification Robot
  Software.

Dementieva et al. 2002