Allergen of Japanese Cedar Pollen, Cryj2, in Escherichia coli

Transcription

1 Chaperone Coexpression Plasmids: Differential and Synergistic Roles of DnaK-DnaJ-GrpE and GroELGroES in Assisting Folding of an Allergen of Japanese Cedar Pollen, Cryj2, in Escherichia coli K. Nishihara et al. (1998) Appl. Environ. Microbiol. 64:

2 Outline 1. Introduction 2. Objectives 3. Results 4. Summary 5. Take-home message 6. Discussion and current questions

3 1.1 Introduction - Production of recombinant proteins in E. coli problem: inability of proteins to reach their native conformation rapid degradation aggregation of folding intermediates example: Cryj2 (major allergen of Japanese cedar pollen) expression in E. coli: unstable and rapidly degraded protein

4 aim: ensure the production of an active and stable recombinant proteins ( preventing it from degradation or aggregation) approach: coexpression of molecular chaperones

5 1.2 Introduction: Molecular chaperones synthesis: under positive control of the heat shock 32 transcription factor σ principle: σ 32 encoded by rpoh gene heat-shock induction (signal for high level of denatured proteins) binding to RNA-Pol (website consulted: ) enhanced transcription of heat shock genes (coding for e.g. chaperones and ATP-dependent proteases)

6 two major chaperone teams in E. coli: a) DnaK-DnaJ-GrpE: principle: FU Hartl (2002) Science 295; 1852 maintains nascent or other preexisting proteins in unfolded states

7 b) GroEL-GroES: principle: interacts with partially folded polypeptides assists in additional folding Prof. Dr. B. Westermann; VL Zellbiologie ; SoSe 2011 functions of chaperone systems: facilitation of -/ assistance in correct protein folding labelling of not properly folded proteins for degradation

8 2. Objectives controlled coexpression of the two major E. coli chaperone teams (DnaK-DnaJ-GrpE and GroEL-GroES) under a variety of conditions in E. coli wild type and in several mutant E. coli strains effects of controlled chaperone coexpression on folding, stability and aggregation of the reporter protein Cryj2

9 3.1 Experiment A: Involvement of the chaperones in expression of Cryj2 I. Expression of Cryj2 in chaperone mutants construction of the wild type (control): transformation of a Cryj2-expressing plasmid into E. coli cells construction of chaperone mutants: transformation of a Cryj2-expressing plasmid into a set of temperature-sensitive E. coli mutants growth at 30 C and induction with IPTG

10 Result: DnaK and dnaj mutants a) SDS-PAGE and immunoblotting of whole-cell protein: significant increase in amount of Cryj2

11 b) SDS-PAGE and immunoblotting of soluble and insoluble fractions: significant increase in amount of insoluble (aggregated) Cryj2

12 c) determination of stability of Cryj2 in vivo: slower degradation of Cryj2 increase in stability of Cryj2 conclusion: non-functional DnaK and DnaJ stabilize not properly folded Cryj2 leading to aggregations DnaK and DnaJ are required for efficient folding of Cryj2 and for labelling of misfolded Cryj2 for degradation

13 Results: groel and groes mutants a) SDS-PAGE and immunoblotting of whole-cell protein: significant decrease in amount of Cryj2

14 b) SDS-PAGE and immunoblotting of soluble and insoluble fractions: relatively small amount of aggregated protein

15 c) determination of stability of Cryj2 in vivo: faster degradation of Cryj2 decreased stability of Cryj2 conclusion: non-functional GroEL and GroES destabilize Cryj2 leading to relatively little aggregation GroEL and GroES stabilize and prevent folded Cryj2 from degradation

16 II. Expression of Cryj2 in ΔrpoH mutant transformation of Cryj2-expressing plasmid into ΔrpoH mutant (lacking heat shock transcription factor σ 32 ) drastical reduction of the level of all cytoplasmatic chaperones and ATP-dependent proteases growth at 20 C and induction with IPTG

17 Results: SDS-PAGE and immunoblotting... of whole-cell protein: increase in amount of Cryj2 after fractionation by centrifugation: mainly insoluble Cryj2-form

18 determination of stability of Cryj2 in vivo: significant slow degradation of Cryj2 (even slower than in previous experiment with chaperone mutants) conclusion: chaperones are involved in correct Cryj2 folding the second important class of heat shock proteins (ATPdependent proteases) has an additional effect on production of native Cryj2

19 3.2 Experiment B: Effect of coexpression of the chaperone teams on Cryj2 stability transformation of a Cryj2expressing plasmid and a of the chaperone-expressing plasmid pg-kje6 into E. coli wild type cells induction of synthesis of the two chaperone teams through increasing concentrations of Larabinose and tetracycline respectively

20 Results: coexpression of DnaK-DnaJ-grpE a) SDS-PAGE of chaperone team 1: increased amounts of DnaK, DnaJ and grpe with increasing concentrations of added Larabinose

21 b) SDS-PAGE and immunoblotting of whole-cell protein Cryj2 and of soluble and insoluble fractions: increasing amount of Cryj2 with increasing concentration of Larabinose most Cryj2 in soluble form

22 b) determination of stability of Cryj2 in vivo: 10-fold excess of DnaK, J, GrpE 5-fold stabilization of Cryj2 conclusion: increased coexpression of both chaperone teams stabilizes Cryj2 to comparable extents without causing appreciable aggregation

23 3.3 Experiment C: Effect of coexpression of the chaperone teams on Cryj2 aggregation background: expression of Cryj2 in ΔrpoH mutants (reduced level of chaperones) leads to high amount of aggregated Cryj2 forms question: May coexpression of either or both chaperone teams prevent aggregation in ΔrpoH mutants?

24 I. Coexpression of chaperone teams at 20 C ΔrpoH mutant containing a Cryj2-expressing plasmid and a chaperone team 1-expressing plasmid (and a chaperone team 2-expressing plasmid respectively) growth at 20 C and induction of coexpression through increasing concentrations of L-arabinose and tetracycline respectively

25 Results: SDS-PAGE and immunoblotting chaperone team 1: modest chaperone coexpression sufficient to prevent aggregation of Cryj2 chaperone team 2: greater chaperone coexpression required to prevent aggregation of Cryj2

26 conclusion: coexpression of one chaperone team alone prevents aggregation and reduces the requirement for the other chaperone team

27 II. Coexpression of chaperone teams at 30 C ΔrpoH mutant containing a Cryj2-expressing plasmid and the chaperone-expressing plasmid pg-kje6 growth at 30 C and induction of coexpression

28 Results: SDS-PAGE and immunoblotting coexpression of chaperone teams alone does not prevent Cryj2 aggregation coexpression of both chaperone teams together prevents aggregation

29 conclusions: coexpression of both chaperone teams needed for effective protection against aggregation chaperone teams play synergistic roles

30 4. Summary both chaperone teams are in distinct but cooperative ways critically involved in Cryj2 folding despite the distinct roles in protein folding, excess coexpression of chaperone teams 1 and team 2 respectively has a similiar effect on stabilization of native Cryj2 in wild type cells the two chaperone teams play synergistic and partially compensatory roles in preventing aggregation of Cryj2 in rpoh mutants at certain temperatures

31 5. Take-home message The study of controlled chaperone coexpression and its effect on folding, stability and assembly of proteins in wild-type bacteria as well as in mutant hosts provides a useful approach to improve the production of recombinant proteins.

32 6. Discussion 1. Welche beiden wichtigen Proteinklassen stehen unter der 32 σ Kontrolle von? 2. Wie kann man lösliche von unlöslichen (aggregierten) Proteinen trennen?