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Co-solvents as stabilizing agents during heterologous overexpression in Escherichia coli - application to chlamydial penicillin-binding protein 6.

Otten C, De Benedetti S, Gaballah A, Bühl H, Klöckner A, Brauner J, Sahl HG, Henrichfreise B - PLoS ONE (2015)

Bottom Line: Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies.To solve this problem use of chaperones or in vitro refolding procedures are the means of choice.Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."

View Article: PubMed Central - PubMed

Affiliation: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.

ABSTRACT
Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."

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Application of the co-solvent assisted method to PBP6Cp.(a) Structure of PBP6Cp. Domains, motifs, signal peptides (SP), transmembrane domains (TM), leader peptide sequence for transportation into the periplasm (OmpA) and chromatography affinity tag (Strep) are depicted (SP and TM were predicted by Signal P and TMHMM [30,31]. The modified PBP6Cp (42.95 kDa) which lacks the native SP and TM domain was overproduced, purified and tested for DD-carboxypeptidase activity. (b) Results from overexpression pretest (western blot), (c) co-solvent screen western blot), and (d) betaine-assisted purification (Coomassie stain) of PBP6Cp. The optimal expression conditions (E. coli C43(DE3), 4h of induction at 25°C) and co-solvent (betaine) determined in the overexpression pretest and co-solvent screen, respectively, were used in an up-scaled culture to overproduce soluble PBP6Cp for the first time. Pre: pre induction, S: soluble fraction, P: pellet fraction, w/o: without the addition of co-solvent, Glc: glucose, Suc: sucrose, Tre: trehalose, Lac: lactose, Gly: glycerol, Man: mannitol, Sor: sorbitol, Bet: betaine, His: histidine. BCCP (21.5kDa): biotin carboxyl carrier protein from E. coli, a common contamination of strep-tagged proteins (biotinylated protein binding to strep-tactin which can be removed by complexation with avidin from hen egg white [12]).
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pone.0122110.g002: Application of the co-solvent assisted method to PBP6Cp.(a) Structure of PBP6Cp. Domains, motifs, signal peptides (SP), transmembrane domains (TM), leader peptide sequence for transportation into the periplasm (OmpA) and chromatography affinity tag (Strep) are depicted (SP and TM were predicted by Signal P and TMHMM [30,31]. The modified PBP6Cp (42.95 kDa) which lacks the native SP and TM domain was overproduced, purified and tested for DD-carboxypeptidase activity. (b) Results from overexpression pretest (western blot), (c) co-solvent screen western blot), and (d) betaine-assisted purification (Coomassie stain) of PBP6Cp. The optimal expression conditions (E. coli C43(DE3), 4h of induction at 25°C) and co-solvent (betaine) determined in the overexpression pretest and co-solvent screen, respectively, were used in an up-scaled culture to overproduce soluble PBP6Cp for the first time. Pre: pre induction, S: soluble fraction, P: pellet fraction, w/o: without the addition of co-solvent, Glc: glucose, Suc: sucrose, Tre: trehalose, Lac: lactose, Gly: glycerol, Man: mannitol, Sor: sorbitol, Bet: betaine, His: histidine. BCCP (21.5kDa): biotin carboxyl carrier protein from E. coli, a common contamination of strep-tagged proteins (biotinylated protein binding to strep-tactin which can be removed by complexation with avidin from hen egg white [12]).

Mentions: The PBP6Cp encoding gene dacF from Chlamydia pneumoniae strain GiD [10] was amplified by PCR using primer PBP6ΔSPΔTM_for_Cp and PBP6ΔSPΔTM_rev_Cp (Table A in S1 File) and cloned into pASK-IBA2C (IBA, Germany) resulting in vector pASK-IBA2C_PBP6Cp (Table A in S1 File) that allows for periplasmic expression of PBP6. The sequences coding for the intrinsic N-terminal signal peptide and the C-terminal transmembrane domain was removed and the gene was N-terminally fused to leader peptide OmpA and a Strep tag was added to the C-terminus (Figs 1b and 2a).


Co-solvents as stabilizing agents during heterologous overexpression in Escherichia coli - application to chlamydial penicillin-binding protein 6.

Otten C, De Benedetti S, Gaballah A, Bühl H, Klöckner A, Brauner J, Sahl HG, Henrichfreise B - PLoS ONE (2015)

Application of the co-solvent assisted method to PBP6Cp.(a) Structure of PBP6Cp. Domains, motifs, signal peptides (SP), transmembrane domains (TM), leader peptide sequence for transportation into the periplasm (OmpA) and chromatography affinity tag (Strep) are depicted (SP and TM were predicted by Signal P and TMHMM [30,31]. The modified PBP6Cp (42.95 kDa) which lacks the native SP and TM domain was overproduced, purified and tested for DD-carboxypeptidase activity. (b) Results from overexpression pretest (western blot), (c) co-solvent screen western blot), and (d) betaine-assisted purification (Coomassie stain) of PBP6Cp. The optimal expression conditions (E. coli C43(DE3), 4h of induction at 25°C) and co-solvent (betaine) determined in the overexpression pretest and co-solvent screen, respectively, were used in an up-scaled culture to overproduce soluble PBP6Cp for the first time. Pre: pre induction, S: soluble fraction, P: pellet fraction, w/o: without the addition of co-solvent, Glc: glucose, Suc: sucrose, Tre: trehalose, Lac: lactose, Gly: glycerol, Man: mannitol, Sor: sorbitol, Bet: betaine, His: histidine. BCCP (21.5kDa): biotin carboxyl carrier protein from E. coli, a common contamination of strep-tagged proteins (biotinylated protein binding to strep-tactin which can be removed by complexation with avidin from hen egg white [12]).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388811&req=5

pone.0122110.g002: Application of the co-solvent assisted method to PBP6Cp.(a) Structure of PBP6Cp. Domains, motifs, signal peptides (SP), transmembrane domains (TM), leader peptide sequence for transportation into the periplasm (OmpA) and chromatography affinity tag (Strep) are depicted (SP and TM were predicted by Signal P and TMHMM [30,31]. The modified PBP6Cp (42.95 kDa) which lacks the native SP and TM domain was overproduced, purified and tested for DD-carboxypeptidase activity. (b) Results from overexpression pretest (western blot), (c) co-solvent screen western blot), and (d) betaine-assisted purification (Coomassie stain) of PBP6Cp. The optimal expression conditions (E. coli C43(DE3), 4h of induction at 25°C) and co-solvent (betaine) determined in the overexpression pretest and co-solvent screen, respectively, were used in an up-scaled culture to overproduce soluble PBP6Cp for the first time. Pre: pre induction, S: soluble fraction, P: pellet fraction, w/o: without the addition of co-solvent, Glc: glucose, Suc: sucrose, Tre: trehalose, Lac: lactose, Gly: glycerol, Man: mannitol, Sor: sorbitol, Bet: betaine, His: histidine. BCCP (21.5kDa): biotin carboxyl carrier protein from E. coli, a common contamination of strep-tagged proteins (biotinylated protein binding to strep-tactin which can be removed by complexation with avidin from hen egg white [12]).
Mentions: The PBP6Cp encoding gene dacF from Chlamydia pneumoniae strain GiD [10] was amplified by PCR using primer PBP6ΔSPΔTM_for_Cp and PBP6ΔSPΔTM_rev_Cp (Table A in S1 File) and cloned into pASK-IBA2C (IBA, Germany) resulting in vector pASK-IBA2C_PBP6Cp (Table A in S1 File) that allows for periplasmic expression of PBP6. The sequences coding for the intrinsic N-terminal signal peptide and the C-terminal transmembrane domain was removed and the gene was N-terminally fused to leader peptide OmpA and a Strep tag was added to the C-terminus (Figs 1b and 2a).

Bottom Line: Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies.To solve this problem use of chaperones or in vitro refolding procedures are the means of choice.Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."

View Article: PubMed Central - PubMed

Affiliation: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.

ABSTRACT
Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."

Show MeSH
Related in: MedlinePlus