Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

Abstract

To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJGrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production in E. coli.Recombinant proteins produced in Escherichia coli often aggregate or degrade rapidly because of their inability to form correct tertiary structures due to anomalies in protein folding. In some cases, overexpression of molecular chaperones, such as GroEL-GroES and DnaK-DnaJ-GrpE, facilitate protein folding and enhance production of active proteins (16,18). We previously constructed a series of versatile plasmids for controlled expression of these chaperones and described their utility for stabilizing or preventing aggregation of certain recombinant proteins (9). However, these plasmids are not always effective; there are many instances in which production of active proteins is not improved by the chaperones and seems to require additional factors or conditions. Thus, we examined new chaperonelike factors, including E. coli trigger factor (TF), whose role in protein folding was implicated by the results of several in vitro experiments. This 50-kDa protein exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity and has a domain structure that is conserved in the FK506-binding protein family (14,17). TF was originally identified as a protein that can bind to certain precursor proteins and facilitate their transport into membrane vesicles (1). The results of subsequent studies, however, failed to support hypothesis that TF plays a role in protein transport (2) and instead suggested that it may play a role in protein folding because of its association with nascent polypeptides, as well as the 50S ribosome (3, 17). Moreover, TF was found to be associated with GroEL and can strengthen GroEL-substrate binding to facilitate protein folding (5) or degradation (6).In this study, we constructed plasmids with which we could easily manipulate the production of TF, alone or together with GroEL-GroES chaperones, and examined the effects of coexpression of these molecules on the production of several recombinant proteins. The target proteins employed...

Soluble Expression of Recombinant Human Smp30 for Detecting Serum Smp30 Antibody Levels in Hepatocellular Carcinoma Patients

“…Because successful chaperone co-expression to improve solubility is a trial-and-error process that can not be predicted from properties of proteins (Baneyx et al, 2004;Hoffmann et al, 2004), we explored nine combinations of chaperones four chaperones with pET30a-SMP30 and five chaperones with pColdIII-SMP30, and found that SMP30 was rescued from aggregation by co -expression of chaperones pKJE7, pG-Tf2 and pTf16, especially pTf16. This result was consistent with other studies (Nishihara et al, 2000;Moonsuk et al, 2010).…”

Efficacy of heterologous and homologous heat shock protein 70s as protective agents to Artemia franciscana challenged with Vibrio campbellii

“…Induction of Hsp70 proteins in E. coli strains E. coli strains YS1, YS1 and A native were grown at 37 C for 24 h on LB agar and then to log phase in LB broth by incubation at 37 C. V. campbellii strain LMG 21363 stored in 40% glycerol at À80 C were grown at 28 C for 24 h on marine agar and then to log phase in marine broth 2216 (Difco Laboratories, Detroit, Mich.) by incubation at 28 C. Overproduction of Artemia Hsp70 protein in A native cells was stimulated by adding different doses of L-arabinose (0, 0.5, 1, 2, and 4 mg ml À1 ) for a fixed time (1 h). Subsequently, L-arabinose at 0.5 mg ml À1 , which gave the best induction in the dose-response experiment, was tested for different time intervals (1, 2, 3 and 4 h) [18]. Maximum production of Artemia Hsp70 in A native cells was obtained at 0.5 mg ml À1 L-arabinose for 4 h, as obtained for YS2 cells overproducing DnaK [13].…”

Analysis of σ 32 mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response

“…For chaperone overexpression experiments, chromosomal P ara -groEL/S (32) or P A1/lacO-1 -dnaK/ J-lacI q (15) was transduced into CAG48238 to obtain CAG48239 or CAG48275, respectively. For Table 1, derivatives of CAG48239 carrying a plasmid-encoded rpoH mutations and a pACYC184-based plasmid pGro11 (P tet -groESL) (33) were grown in LB medium containing L-arabinose (0.2%) at 30°C to obtain the WT level of GroEL/S; rpoH was induced by the addition of 1 mM IPTG, and GroEL/S was overexpressed by the addition of 25 ng/ml anhydrotetracycline. For DnaK/J overexpression, derivatives of CAG48275 carrying plasmid-encoded rpoH mutations were grown in 5 M IPTG at 30°C to achieve the WT level of DnaK/J from the chromosomal P A1/lacO-1 -dnaK/JlacI q locus.…”

“…For DnaK/J overexpression, derivatives of CAG48275 carrying plasmid-encoded rpoH mutations were grown in 5 M IPTG at 30°C to achieve the WT level of DnaK/J from the chromosomal P A1/lacO-1 -dnaK/JlacI q locus. DnaK/J and 32 were simultaneously overexpressed by the addition of 1 mM IPTG; alternatively, DnaK/J was overexpressed from another compatible plasmid pKJE8 (P aradnaK/J/grpE) by the addition of L-arabinose (33). The differential rate of ␤-galactosidase synthesis was determined over a 2-h time course immediately after chaperone induction.…”