Microsoft word - protocol_expressionscreen.doc

Gunter Stier, Heidelberg: E.coli expression, CPEP-09, KBC Umeå Miniexpression screen
Part 1: Fast transformation

Ready circular plasmids can be transformed much faster than ligations, especially in the high
competent [Inoue, et al.] cells. Transformation efficiency drops, but still good enough. Plating on
sectors saves a lot of plates and is good enough for screening. BL21 (DE3) pLysS strain is based
on a very stable low copy number plasmid, visible as Chloramphenicol AcetylTransferase band at
around 25 kD on the protein gel. You don’t need to go on double resistance usually. Only then the
ON is started with both antibiotics.
The tested plasmids are numbered and represent cases with the following characteristic features:
• M-series vectors with colored marker proteins • M52 (dsbA) with an extracellular Ig domain (S-S bridge) • M-series with short “Scottish” peptide • Recombinant Tev protease • ProteinX with many rare codons in the cDNA fused to C terminal GFP
1. Thaw competent BL21[DE3] or pLysS version on ice.
2. Add 50 ul cells to 10 ng (1 ul of QuiaprepDNA) of your plasmid.
3. Inoculate 15 min on ice.
4. Heat shock 1 min 420 C.
5. Spin cells and discard most of SN.
6. Plate 20 ul of each pellet on 16 sectors of prewarmed plates by
setting drops and leave them on bench for drying.
(alt. plus 1 ml SOC and 15 min 370 C).
Gunter Stier, Heidelberg: E.coli expression, CPEP-09, KBC Umeå
Part 2: Expression screen

Ready circular plasmids can be transformed much faster than ligations, especially in the high
competent [Inoue, et al.] cells. Transformation efficiency drops, but is still good enough. Plating
on sectors saves a lot of plates and is good enough for expression screening. The BL21(DE3)
pLysS strain is based on a very stable low copy number plasmid, visible as Chloramphenicol
AcetylTransferase band at around 25 kD on the protein gel. This is why you don’t need to go on
double resistance for the plating of the transformants. Only then, when you pick the colonies next
day, the initial ON is started with both antibiotics. If you use this strain you have to induce much
longer, since the promoter is not as leaky as in the BL21(DE3).
1. Plate single colonies on agar plate with antibiotics and grow ON or start with fresh
transformants.
2. Scrape colonies off the plate with a full 10 ul loop and directly induce in 1 mM IPTG/LB
(Full 10 ul loop cells in 200 ul LB/IPTG).
• Alternatives: Both are more reliable, but take more time
o Start 10 ml culture from an ON inoculation, grow OD 0.6 and induce
o Start single colony in an autoinduction medium and grow via vigorous
3. Shake 30 min - 2 hr (pLysS 3 -12 hrs) 37 0 C or desired temperature.
4. Spin cells 30 sec.
5. Dissolve cell pellet in 0.5 ml of lysis buffer.
6. Add 0.5 ml lysozyme/DNAseI solution in lysis buffer.
7. Sonicate each 10-30 sec with a microtip (avoid foaming).
8. Spin 5 min in 2 ml eppendorf tubes at 40 C.
9. Meanwhile prepare Microspin columns with 30-50 ul equilibrated Ni - agarose and
place them on 8 ml Falcon tubes.
10. Decant SN on Microspin column and reload follow-through 3 times.
11. Wash with 1 ml each wash1/wash 2/wash3 .
12. Elute with 150 - 200 ul elution buffer.
• Measure eluate in a protein stain assay
• Run SDS gel
13. Take 50 ul sample and add 5 ul Tev protease stock solution.
14. Digest 15-30 min at RT.
15. Run analytical gel.
Gunter Stier, Heidelberg: E.coli expression, CPEP-09, KBC Umeå Buffers:
Lysis buffer & Wash 1
• 20 mM Tris pH 8.0
• Lysis buffer minus NP40
• 10 mM Imidazole pH 8.0
• 150 mM NaCl
• 0.2% NP-40
• Wash 2 / ca. 1 M NaCl
• 2 mM Mercaptoethanol

Elutionbuffer
• 1 uM PEFAC
• Wash 2 / 330 mM Imidazole
+ Lysozyme1 mg/ml
/DNAseI 5 μg/ml final conc. Only add to
lysates from a small stock.


Example:
Columns:

Protein assay:

Gunter Stier, Heidelberg: E.coli expression, CPEP-09, Interesting samples for a analytical SDS-gel:
Supernatant:
proteins
Insoluble proteins (Inclusion bodies), cell debris
Flow through: F
Proteins not binding. If the His-tag is hidden
(structural reasons) the target protein won’t
bind
1 /2:M10 3:M11 4:Mcoex 5:M12 6:M20 7 M30 8:M41 9:M52 13:M60 + Tev protease

Source: http://babel.ucmp.umu.se/cpep/web_content/pdf/protocols/protocol_Expressionscreen.doc.pdf