CNRS - AIX MARSEILLE UNIV: UMR7257

Home page > en > Facilities > Structural Biology > Cloning, expression in bacteria, purification of recombinant (...) > Cloning, expression in bacteria, purification of recombinant (...)

Cloning, expression in bacteria, purification of recombinant proteins

Heads Renaud VINCENTELLI

Introduction

JPEG - 103.9 kb
The AFMB pipeline

The cloning, expression and protein purification facility of the AFMB (IBiSA) is opened to customers since 2003 and is linked to the protein characterization and crystallography facilities of the lab.

References

  • Vincentelli R, Bignon C, Gruez A, Canaan S, Sulzenbacher G, Tegoni M, Campanacci V, Cambillau C (2003) Acc Chem Res 36 165-172
  • Sulzenbacher G, Gruez A, Roig-Zamboni V, Spinelli S, Valencia C, Pagot F, Vincentelli R, Bignon C, Salomoni A, Grisel S, Maurin D, Huyghe C, Johansson K, Grassick A, Roussel A, Bourne Y, Perrier S, Miallau L, Cantau P, Blanc E, Genevois M, Grossi A, Zenatti A, Campanacci V, Cambillau C (2002) Acta Crystallogr D Biol Crystallogr 58 2109-2115

We are using our own high-throughput strategies and methods which were developped and validated for our structural genomics projects since 2001 (ASG, X-TB, SPINE, SPINE II, EMEP, MEPNET, VIZIER, VENOMICS (www.venomics.eu)...). We have the state-of-the-art equipment and the method developments are constant. In the past five years we have worked on several thousands of proteins for our customers and collaborators.

References

  • Vincentelli R, Cimino A, Geerlof A, Kubo A, Satou Y, Cambillau C (2011) Methods 55 65-72
  • Graslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schutz A, Heinemann U, Yokoyama S, Bussow K, Gunsalus KC (2008) Nat Methods 5 135-46

Next : Overall Strategy

Strategy

Overall strategy

After cloning the gene of interest in one or several expression vectors, we optimize the level of soluble expression at analytical scale on a Tecan robot playing with various culture conditions ans protein fusions. We do a 1-5 litres culture to purify the protein in suitable quantities for the customers only once we have found optimal conditions for the soluble expression.

JPEG - 55.4 kb
Recombinant protein production optimisation of protein soluble expression on the automated expression screening

If we can only detect the protein in inclusion bodies we do a renaturation screen or modify the protein sequence. All the proteins are controlled (Capillary electrophoresis and Mass spectrometry) before they are shipped to the customer with the report.

Previous : Introduction / Next : Cloning

Cloning

We mainly use E. Coli as an expression host. We use the Gateway technology (Life Tech ) in 96 well format but other cloning methods are possible. The cloning is done using a template supplied by the customer or a synthetic gene. The clones are sequenced and sent to the customers.

JPEG - 82.2 kb
Cloning with the Gateway Technology (Life Tech)

We have more than ten custom made vectors for E. Coli.

Previous : Overall Strategy / Next : Expression

Expression

Expression and solubility screening

We and others have demonstrated the impact of the culture conditions and plasmid constructs on the level of expression and solubility. To be able to quickly screen for optimal conditions we have developped and published a high throughput expression screen protocol based on an automated dotblot. We are able to work on up to 1152 cultures/week with 1 ml culture in deep-well 96.

References

  • Berrow NS, Bussow K, Coutard B, Diprose J, Ekberg M, Folkers GE, Levy N, Lieu V, Owens RJ, Peleg Y, Pinaglia C, Quevillon-Cheruel S, Salim L, Scheich C, Vincentelli R, Busso D (2006) Acta Crystallogr D Biol Crystallogr 62 1218-26
  • Vincentelli R, Canaan S, Offant J, Cambillau C, Bignon C (2005) Anal Biochem 346 77-84
  • Vincentelli R, Coutard B, Cambillau C (2006). Trends in drug discoveries, 2, 16-18.
  • Vincentelli R, Cambillau C (2005). Tecan Journal, 2, 20-21
JPEG - 47.4 kb
The automated expression screening (Crédit : Perrin, CNRS)

The two TECAN Evo 200 robots are used for the cloning, the expression screening, the refolding screen… And all kind of custom made assays in 96 well format.

JPEG - 76.8 kb
Protein solubility level quantified by automated dotblot on 1152 cultures in parralel

Since 2010, in most applications, the dotblot has been replaced by capillary electrophoresis detection on a Caliper GX II (Perkin Elmer). This gives access to the protein concentration, purity and molecular weight in 96/384 with a speed and sensitivity comparable with the dotblot.

JPEG - 65.1 kb
Protein solubility level quantified by capillary electrophoresis in 96/384

The analysis of our statistics (based on thousands of cultures) on the impact of the culture conditions and plasmid constructs on the level of solubility allowed us to streamline our protocol to offer a simpler and more efficient procedure than before. If this unique protocol fails, we screen culture conditions and fusion in a logical way.

JPEG - 67.2 kb
Simplified expression screening protocol

Previous : Cloning / Next : Refolding of inclusion bodies

Refolding

Refolding of inclusion bodies

If we detect only the protein in inclusion bodies we use our own home made renaturation kit to try to refold the proteins and purify them in native conditions. We can also purify the proteins in a denatured form (to produce antibodies for instance).

References

  • Vincentelli R, Canaan S, Campanacci V, Valencia C, Maurin D, Frassinetti F, Scappucini-Calvo L, Cambillau C, Bignon C (2004) Protein Sci 13 2782-2792
  • Vincentelli R (2007) in Expression Systems (Michael Dyson and Yves Durocher Eds), Scion publishing.

Previous : Expression / Next : Purification

Purification

JPEG - 116.7 kb
[enAKTA Xpress (GE Healthcare) Crédit Perrin, CNRS)

The pure proteins can then be transfered on the protein characterization and crystallogenesis facilities of the lab.

Custom development of tests in 96 or 384 format

We can develop various custom HTP assays. Once validated, if possible, they enrich the portfolio of protocols and can be used by customers and collaborators.

We have recently developped two in vitro tests; a protein-DNA interaction assay (HTP SELEX 96) and a protein-protein interaction assay (HTP Pull-Down or HTP Hold-up 96/384). These protocols will be accessible once published.

Contact: R. Vincentelli (renaud.vincentelli afmb.univ-mrs.fr)

© AFMB UMR7257  W3C validation