1

Typical lab spirit

What to prepare :What to prepare :

• Order primer ( following slides) : Discuss with organizersBest including NcoI _ Acc65I sites (see also following slides)

(also possible NcoI - NotI, or BpiI - Acc65I or NotI)

• Run PCR• Cut PCR fragment• Clone in basic vector ( Get from course organizers)• Recut and prepare restriction fragment with NcoI/Acc65I

We can start with anything you have in preparation, but best if you do the following steps before the course starts :

NcoI Acc65Iyour Gene (insert)

Cloning in multiple vectors

Vector alone Same vector with fragments

Ca 50 colonies > 1000 colonies

2

Transformation intoexpression strain

Results: Autoinduction and preparation ofprotein samples

5 - 10 OD stable ON expression at 24 deg > 50 expression clones screened

Autoinduction:• easy handling • more material

• wet weight • protein for miniscreen

Group C :3 out of 4 targetsare soluble and well cleaved with Tev

Next step:

• Upscaling• N15 sample

Preparation:

• Material from 1 ml ON autoinduced• 50 ul Ni column• 250 ul Eluate• 50 ul Test ON digest with 5 ul Tev

Midiprotein preps A &B

3

2nd Ni-column

Complete cleaved coloured protein should be all inthe flow after 2nd Ni column - yes it is !!

No colour left

Cpep: Group AGroup A: Daniel, Christian & Jagan• Sequence analysis, internal restriction sites, primer design• PCR cloning to circumvent internal site problem ?• Cloning from source clones in series via partial digested fragment• Miniexpression screen• Protein midiprep• Autoinduction mediumSpecial subjects:

• Coexpression of proteins in different systems

Special seminar : Coexpression in two-plasmidsystems, Multicistronic vectors, Baculovirus vectorsand coexpression (Multibac-system)

Group A

3 systems:pACYC /dicistronic/combinations

Coexpression

24 x 3 clones4 x 344

Soluble/purifiedcandidates

ExpressionsSubclones/vectorsSource clonesPCR

Cloning despite internal sitesPCR and cut and isolate 2 fragments:

• NcoI / XbaI fragment• XbaI / Acc65I fragment

NcoINcoIXbaI Acc65I

NcoI XbaI

Acc65IXbaI

NcoI XbaI Acc65I

Ligate inExpressionvector

4

Cloning of subfragments fromsource clone

Acc65IXbaI

NcoI XbaI Acc65I

NcoIXbaI

NcoIXbaI NcoI

religate

Group A: Miniscreen: Jagan

Result:• Low protein production• Many subbands

• Translational stops ?• Rare codons ?• Instability and degradation

• Aberrant running behaviour • Unstructured areas• Coiled coils

Next step:

• Rosetta as expression strain• Check Total/SN/pellet/eluates • New subdomain constructs of possible inteaction areas• Low temperature induction• Coexpression strategies with partners and subdomains

Miniscreen: Group A Coexpression: Diplasmid

2 plasmids:• pET21d• pACYCT7 His

x

Cotransformation

5

Group A: Dicistronic (oligomethod)

2 cistrons:• H6ZZ GFP (bound)• trx1b Dan (? Insoluble alone)

rbs oligoT7rev

x

Coligate in any pET

Group A: Christian screen

Result:• Low protein production• Good His-tag versions, but low yield •Many subbands

• Translational stops ?• Rare codons spotted on gene - consecutive rare Arg• Instability and degradation

Next step:

• Rosetta as expression strain• Check Total/SN/pellet/eluates

Group A: Low temperature induction Group A: Protein preparation

6

Group A: Protein preparation Cpep: Group BGroup B: Helena, Martin & Sajna• Primer design• PCR of ORF• Source clone• Subcloning into different vectors• Autoinduction medium• Miniexpression screen• Protein preparationSpecial subjects:

• T7 RNA polymerase purification and Akta• Tev protease : Screening for active domain

Special seminar : Cell free expression,Nanodiscs, Cell free expression protocol formembrane proteins, ATPase production in-vitro

Group B: Test of expression and proteinpreparation of upscaled proteins samples

Group B: T7 RNA polymerasepurification

Result from 300 ml autoinduction :• Good protein production• Ni column• Mono Q• Gelfiltration• Stored -70 deg

Next step:

• Speed up protein preparation• Upscale expression volume• Assay protein activity in

• RNA synthesis• Cell free expression

7

Group B: ORF of unknownfunction

Analysis: • Expression of fusions with ORF - trx in pellet• MBP is produced without ORF and purified over Ni-column

Next step:

• No tag version• N and c-His version• Low temperature induction• Bioinformatics to construct other versions • Baculovirus expression

Tev protease variants andtheir activity

Tev protease deletion variants are cloned in 3 vectors:• GST 1a incl.Tev site - should cleave itself in-vivo• His tag - should be active in substrate cleavage• GST-dir - should be active in substrate cleavage

1. Parallel cloning2. Expression test3. Purification 4. Acitivity test

Biorad assay of eluates

Tev protease variants andtheir activity

Tev protease deletion variants are cloned in 3 vectors:• GST 1a incl.Tev site - should cleave itself in-vivo• His tag - should be active in substrate cleavage• GST-dir - should be active in substrate cleavage

Acitivity test

Active variant cleavesSubstrate as His tag protein

Cpep: Group C

Group C: Lotta, Xu & Dharmesh• Primer design• Subcloning in expression vectors• Miniexpression screen • Autoinduction• Expression cloning for small peptides• Proteinpreparation for NMRSpecial subjects:

• Domains and screening for soluble constructs with fusion proteins

Special seminar : Library screen, GPCR engineering, Purification of membrane protein complexes

8

Group C: Aaron miniscreen

Soluble candidates

Preparation:

• Material from 1 ml ON autoinduced• 50 ul Ni column• 250 ul Eluate• 50 ul Test ON digest with 5 ul Tev

Group C: Lotta 1 Jagan

Group C: Lotta 2 smallpeptides and tags

Group C: His versus fusion: Small peptides arenot produced with Histag alone

9

Cpep: Group D

Group D: Oanh, Geetanjali & Ummehan• Primer design• Subcloning in expression vectors• Miniexpression screen • Autoinduction• Protein preparationSpecial subjects:

• N15 labelling, NMR probes, phosphatase expression

Special seminar:Localization of mRNA in E.coli, MBP fusions Special fusions for membrane proteins (mistic)

Group D: Fast preparation of upscaled N15samples for NMR

Group D: N15 HisGB1 Group D: N15 mGFP

10

Literature presented by groups C&D

Roosild TP, Greenwald J, Vega M, Castronovo S, Riek R, Choe S..NMR structure of Mistic, a membrane-integrating protein for membrane protein expression.Science. 2005 Feb 25;307(5713):1317-21

Nevo-Dinur K, Nussbaum-Shochat A, Ben-Yehuda S, Amster-Choder O. Translation-independent localization of mRNA in E. coli.Science. 2011 Feb 25;331(6020):1081-4.

Hu J, Qin H, Gao FP, Cross TA. A systematic assessment of mature MBP inmembrane protein production: overexpression, membrane targeting and purification. Protein Expr Purif. 2011 Nov;80(1):34-40. Epub 2011 Jun 13.

Hart DJ, Tarendeau F. Combinatorial library approaches for improving solubleprotein expression in Escherichia coli. Acta Crystallogr D Biol Crystallogr. 2006Jan;62(Pt 1):19-26. Epub 2005 Dec 14. Review.

Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, ChaePS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH,Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK. Crystal structure ofthe beta2-adrenergic receptor-Gs protein complex. Nature. 2011 Jul 19;477(7366):549-55.

Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC, Kobilka BK. GPCR engineering yieldshigh-resolution structural insights into beta2-adrenergic receptor function.Science. 2007 Nov 23;318(5854):1266-73. Epub 2007 Oct 25. PubMed PMID: 17962519.

Literature presented by groups A&BBorch J, Hamann T. The nanodisc: a novel tool for membrane protein studies.Biol Chem. 2009 Aug;390(8):805-14.

Matthies D, Haberstock S, Joos F, Dötsch V, Vonck J, Bernhard F, Meier T. Cell-free expression and assembly of ATP synthase. J Mol Biol. 2011 Oct 28;413(3):593-603. Epub 2011 Sep 10.

Birgit Schneider, Friederike Junge, Vladimir A. Shirokov, Florian Durst, Daniel Schwarz, Volker Dötsch, and Frank Bernhard Chapter 11: Membrane Protein Expression in Cell-Free SystemsI. Mus-Veteau (ed.), heterologous Expression of Membrane Proteins, Methods in molecular Biology, vol. 601DOI 10.1007/978-1-60761-344-2_11, © Humana Press, a part of Springer Science + Business Media, LLC 2010

Seeliger MA, Young M, Henderson MN, Pellicena P, King DS, Falick AM, Kuriyan J. High yield bacterial expression of active c-Abl and c-Src tyrosine kinases.Protein Sci. 2005 Dec;14(12):3135-9. Epub 2005 Oct 31.

Tan S, Kern RC, Selleck W. The pST44 polycistronic expression system forproducing protein complexes in Escherichia coli. Protein Expr Purif. 2005 Apr;40(2):385-95.

Tan S. A modular polycistronic expression system for overexpressing proteincomplexes in Escherichia coli. Protein Expr Purif. 2001 Feb;21(1):224-34.

Berger I, Fitzgerald DJ, Richmond TJ. Baculovirus expression system for heterologous multiprotein complexes.Nat Biotechnol. 2004 Dec;22(12):1583-7. Epub 2004 Nov 28.

Imasaki T, Calero G, Cai G, Tsai KL, Yamada K, Cardelli F, Erdjument-Bromage H, Tempst P, Berger I,Kornberg GL, Asturias FJ, Kornberg RD, Takagi Y. Architecture of the Mediator head module.Nature. 2011 Jul 3;475(7355):240-3.