Global Sales Review September 2008

1. NEB Competent Cell Product Portfolio February 2011 Global Sales Review September 2008 Jim Samuelson Gene Expression Division

2. The NEB Competent Cell Product Portfolio For Protein Expression NEB Express NEB Express Iq T7 Express T7 Express Iq T7 Express lysY/Iq T7 Express lysY T7 Express Crystal Shuffle Express Shuffle T7 Express Shuffle T7 Express lysY Shuffle Shuffle T7 Shuffle T7 lysY For Cloning/ Plasmid Isolation NEB 10-beta NEB 5-alpha NEB 5-alpha Iq NEB Turbo dam-/dcm- BL21(DE3) Lemo21(DE3) New Dec 2010 NiCo21(DE3) BL21

3. Cloning strain vs. Protein expression strain • Cloning strain • Essential: High transformation efficiency (>1 x 109) • endA1 Activity of nonspecific endonuclease I is eliminated • for highest quality plasmid preparations • Desirable: recA1 Reduced recombination of cloned DNA • Protein expression strain • Desirable: protease deficient (e.g. Lon, OmpT) • endA1(all “Express” strains carry this mutation) • lacIqand/or lysY function to control basal protein expression

4. Strains For Cloning and Plasmid Isolation NEB 10-beta NEB 5-alpha NEB 5-alpha Iq NEB Turbo dam-/dcm-

5. C3019H/I C3020K NEB 10-beta Competent E. coli • * DH10B™ equivalent • * Reduced recombination of cloned DNA (recA1) rapid growth recA strain • * Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations • * Efficient transformation of methylated DNA derived from eukaryotic sources or unmethylated DNA derived from PCR, cDNA and many other sources [mcrAΔ(mrr-hsdRMS-mcrBC)] • * Resistance to phage T1 (fhuA2) • * Suitable for blue/white screening with cloning vectors expressing the lacZα peptide • (No IPTG required if plasmid does not encode the lacI gene, only X-gal required) • * transformation of large plasmids and BACs • * K12 Strain

6. C2987H/I C2989K NEB 5-alpha Competent E. coli * DH5α™ equivalent * Reduced recombination of cloned DNA (recA1) * Activity of endonuclease I (endA1) eliminated for highest quality plasmid preparations * Free of animal products * Efficient transformation of unmethylated DNA derived from PCR, cDNA and many other sources (hsdR) * Resistance to phage T1 (fhuA2) * Suitable for blue/white screening with cloning vectors expressing the lacZα peptide * K12 Strain

7. NEB 5-alpha IqCompetent E. coli C2992H/I * DH5α™ equivalent * Tight control of expression by laclq allows potentially toxic genes to be cloned * Reduced recombination of cloned DNA (recA1) * Activity of endonuclease I (endA1) eliminated for highest quality plasmid preparations * Free of animal products * Efficient transformation of unmethylated DNA derived from PCR, cDNA and many other sources (hsdR) * Resistance to phage T1 (fhuA2) * Suitable for blue/white screening with cloning vectors expressing the lacZα peptide * K12 Strain

8. C2984H/I C2986K NEB Turbo Competent E. coli • * Tight control of expression by laclq allows potentially toxic genes to be cloned • * Highest growth rate on agar plates - visible colonies 6.5 hours after transformation • * Isolate DNA after 4 hours of culturing a single overnight colony • * Activity of endonuclease I (endA1) eliminated for highest quality plasmid preparations • * Resistance to phage T1 (fhuA2) • * Suitable for blue/white screening with cloning vectors expressing the lacZα peptide • * Suitable for 5 minute transformation protocol with AmpR plasmids • * EcoKr-m-, McrBC- • * K12 Strain

9. NEB Turbo – The Fastest Growing Competent Cells Available • Colonies visible after just 6.5 h. • Available as chemical or electrocompetent cells.

10. NEB Turbo is recA+ • When should this be a concern during cloning experiments? • Cloning vector contains regions of homology to the E. coli chromosome • Cloned DNA fragment contains homology to the E.coli chromosome • Cloned DNA fragment contains repeat elements • Undesired clone recombination is more likely with high-copy vectors • e.g. pUC origin of replication • pUC copy number may be reduced by reducing the growth temperature

11. C2925H dam-/dcm-Competent E. coli • * Dam and Dcm methylation (shown below) is not present in the dam-/dcm-strain • 5’-GATC-3’ 5’-CCWGG-3’ • 3’-CTAG-5’ 3’-GGWCC-5’ • * Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations • * Free of animal products • * T1 phage resistant (fhuA2) • * K12 Strain Note: strain is resistant to chloramphenicol m m m m

12. Protein Expression strains BL21(DE3) Lemo21(DE3) NiCo21(DE3) BL21 NEB Express NEB Express Iq T7 Express T7 Express Iq T7 Express lysY/Iq T7 Express lysY T7 Express Crystal Shuffle Express Shuffle T7 Express Shuffle T7 Express lysY Shuffle Shuffle T7 Shuffle T7 lysY also B strains Notes: “Express” indicates a B strain “T7” or “DE3” indicates compatibility with T7 promoter vectors K-12 strains

13. NEB Express, NEB Express Iq or BL21 C3037 C2530 C2523 • * Deficient in proteases Lon and OmpT • * Do not express the T7 RNA polymerase Applications: protein expression from lac, tac, trc, and araBAD promoters recommended strains for pMAL expression of MBP fusion proteins NEB Express Iq is especially useful for protein expression from vectors without the lacI gene Note:NEB Express Iq is resistant to chloramphenicol

14. BL21(DE3) The most commonly used protein expression strain gene1 = phage T7 RNA polymerase gene UV5 = lacUV5 promoter, repressed by LacI protein induced by IPTG

15. Comparison: BL21(DE3) vs. T7 Express strains BL21(DE3) is truly difficult to make chemically competent T7 Express strains offer approx. 100-fold higher transformation efficiency BL21(DE3) expresses endonuclease I – direct cloning is not advised T7 Express strains are endA1 - direct cloning is an option, isolated plasmid is clean enough for sequencing BL21(DE3)-protein expression from T7 promoters is not tightly controlled

16. T7 expression is not tightly controlled in BL21(DE3) Remedy: co-expression of T7 lysozyme, the natural inhibitor of T7 RNA polymerase BL21(DE3) pLysS = moderate expression of wt T7 lysozyme BL21(DE3) pLysE = high expression of wt T7 lysozyme T7 Express lysY and T7 Express lysY/Iq Expression of K128Y variant of T7 lysozyme

17. Strict control of T7 expression in lysY strains Remedy: co-expression of T7 lysozyme, the natural inhibitor of T7 RNA polymerase lysY is the K128Y variant of T7 lysozyme • controls T7 RNA polymerase expression of protein • Cell envelope integrity is not • compromised (no amidase activity) • lysY is carried by a single-copy mini-F plasmid in T7 Express strains • Antibiotic selection is not necessary Zn K128Y Cheng et al. Proc. Natl. Acad. Sci., USA (1994)

18. Comparison: BL21(DE3) vs. T7 Express strains Non-toxic protein expression minus/ plus inducer Toxic clone transformation IPTG

19. T7 Express Crystal Competent E. coli (High Efficiency) C3022H/I Designed for seleno-methionine labeling of proteins for x-ray crystallography studies metB1 mutation allows user to dictate the level of seleno-methionine incorporation NotI x-ray diffraction pattern NotI crystal

20. Seleno-methionine labeling: 3 methods %labeling genotype • auto-induction in BL21(DE3) and derivatives metB+>90% • F.W. Studier Protein Expr Purif. 2005 May ; 41(1): 207–234. • Suppression of methionine biosynthesis by addition metB+>90% • of hydrophobic amino acid cocktail 3) metB->90% or lower if necessary T7 Express Crystal protocol for regulated or constitutive expression systems (see www.neb.com)

21. Lemo21(DE3) The superior T7 strain for producing: Membrane proteins Secreted proteins periplasm outer membrane inner membrane cytoplasm

22. Most extra-cytoplasmic proteins travel through the SecYEG translocase The “bottleneck” E. coli inner membrane YidC

23. “Tuning Escherichia coli for membrane protein overexpression” Samuel Wagner, Mirjam M Klepsch, Susan Schlegel, Ansgar Appel, Roger Draheim, Michael Tarry Martin Hogbom, Klaas J. van Wijk, Dirk J. Slotboom, Jan O. Persson, and Jan-Willem de Gier 105:14371-14376 (2008) Center for Biomembrane Research Stockholm University Xbrane Bioscience

24. Lemo21(DE3) Tunable T7 expression is achieved by modulating the activityof T7 RNA polymerase, not the amount of polymerase C2528H ”Less is more”

25. Lemo21(DE3)2 plasmid expression system Xbrane Bioscience Stockholm University

26. Protein Expression Protocol example: pET21 construct BL21(DE3) Lemo21(DE3) Transform, select on Amp plates Inoculate starter culture Inoculate expression culture Grow to mid-log: induce with 40-400 uM IPTG Continue growing 3-20 hours Harvest cells Transform, select on Amp, Cam plates Inoculate starter culture Inoculate expression culture, Add 0-2000 uM L-rhamnose Grow to mid-log: induce with 400 uM IPTG Continue growing 3-20 hours Harvest cells Important: IPTG level is not varied Only rhamnose is varied

27. Example of membrane protein expression optimization 100uM IPTG 0 100 500 1000 uM rhamnose 400 uM IPTG 0 100 500 1000 uM rhamnose 400 uM IPTG p8CBD-PhoA expression 22 hr Results Samuelson lab September 2008

28. NiCo21(DE3) -designed for the expression and purification of His-tagged proteins BL21(DE3) derivative: endogenous GlmS is mutated (6His to 6Ala) and 3 host metal binding proteins are tagged with chitin binding domain (CBD) Ni-NTA ↵  target      metal-chelate column chitin column target target  NiCo21(DE3)  Expression of His-tagged target protein Removal of CBD-tagged contaminants by chitin beads

29. NiCo21(DE3) Competent E. coli C2529H

30. + cytoplasmic isomerase/ chaperone SHuffleTM a novel E. coli strain capable of correctly oxidizing proteins oxidizing periplasm oxidizing and reducing cytoplasm SHUFFLE wt+

31. Cys CH2 S S CH2 Cys Disulfide Bond Formation Cys CH2 SH Reduction + 2H+ + 2e- Oxidation SH acceptor CH2 O2 or enzyme catalyst e.g. DsbA Cys

32. disulfide bonds are important for protein folding and stability • found in secreted extracytoplasmic proteins: • e.g. antibodies, hormones, proteases, cytokines

33. Prokaryotic Eukaryotic disulfide bond formation cytoplasm Eukaryotic Endoplasmic Reticulum Gram negative bacterial periplasm dsbA dsbC PDI PDI

34. trx/grx dsbC trx/grx dsbC SHuffle = Origami + cytoplasmic DsbC oxidizing cytoplasm periplasm

35. Available from NEB SHuffle Express SHuffle T7 Express SHuffle T7 Express lysY SHuffle SHuffle T7 Shuffle T7 lysY E. coli B E. coli K12 SHuffle™ Sampler Pack C3032I

36. For licensing information regarding: the SHuffle strains, Nico21(DE3) Lemo21(DE3) or any strain expressing lysY Please contact the NEB business development office Email address: bus.dev@neb.com

37. Additional resources may be found at www.neb.com