Roles and applications of small heat shock proteins in the production of recombinant proteins in Escherichia coli

Proteome profiling of the inclusion body (IB) fraction of recombinant proteins produced in Escherichia coli suggested that two small heat shock proteins, IbpA and IbpB, are the major proteins associated with IBs. In this study, we demonstrate that IbpA and IbpB facilitate the production of recombinant proteins in E. coli and play important roles in protecting recombinant proteins from degradation by cytoplasmic proteases. We examined the cytosolic production, and Tat- or Sec-dependent secretion of the enhanced green fluorescent protein (EGFP) in wild type, ibpAB(-) mutant, and ibpAB-amplified E. coli strains. Analysis of fluorescence histograms and confocal microscopic imaging revealed that over-expression of the ibpA and/or ibpB genes enhanced cytosolic EGFP production whereas knocking out the ibpAB genes enhanced secretory production. This strategy seems to be generally applicable as it was successfully employed for the enhanced cytosolic or secretory production of several other recombinant proteins in E. coli.     

The small heat shock protein IbpA of Escherichia coli cooperates with IbpB in stabilization of thermally aggregated proteins in a disaggregation competent state

The small heat shock proteins are ubiquitous stress proteins proposed to increase cellular tolerance to heat shock conditions. We isolated IbpA, the Escherichia coli small heat shock protein, and tested its ability to keep thermally inactivated substrate proteins in a disaggregation competent state. We found that the presence of IbpA alone during substrate thermal inactivation only weakly influences the ability of the bi-chaperone Hsp70-Hsp100 system to disaggregate aggregated substrate. Similar minor effects were observed for IbpB alone, the other E. coli small heat shock protein. However, when both IbpA and IbpB are simultaneously present during substrate inactivation they efficiently stabilize thermally aggregated proteins in a disaggregation competent state. The properties of the aggregated protein substrates are changed in the presence of IbpA and IbpB, resulting in lower hydrophobicity and the ability of aggregates to withstand sizing chromatography conditions. IbpA and IbpB form mixed complexes, and IbpA stimulates association of IbpB with substrate.     

Escherichia coli small heat shock proteins IbpA/B enhance activity of enzymes sequestered in inclusion bodies

Escherichia coli small heat shock proteins, IbpA/B, function as molecular chaperones and protect misfolded proteins against irreversible aggregation. IbpA/B are induced during overproduction of recombinant proteins and bind to inclusion bodies in E. coli cells. We investigated the effect of DeltaibpA/B mutation on formation of inclusion bodies and biological activity of enzymes sequestered in the aggregates in E. coli cells. Using three different recombinant proteins: Cro-beta-galactosidase, beta-lactamase and rat rHtrA1 we demonstrated that deletion of the ibpA/B operon did not affect the level of produced inclusion bodies. However, in aggregates containing IbpA/B a higher enzymatic activity was detected than in the IbpA/B-deficient inclusion bodies. These results confirm that IbpA/B protect misfolded proteins from inactivation in vivo.     

Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli

To investigate the function of Escherichia coli small heat shock proteins, IbpA and IbpB, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also an ibpAB-disrupted strain. The ibpA-, ibpB- and ibpAB-overexpressing strains were found to be resistant not only to heat but also to superoxide stress. However, the ibpAB-disrupted strain was not more sensitive to these stresses than the wild-type strain. The heat sensitivity of a rpoH amber mutant was partially suppressed by the overexpression of plac::ibpAB. These results suggest that IbpA and IbpB may be involved in the resistances to heat and oxidative stress.     

IbpA/B Small Heat-Shock Protein of Marine Bacterium Vibrio harveyi Binds to Proteins Aggregated in a Cell During Heat Shock

The IbpA and IbpB are 16-kDa Escherichia coli proteins belonging to a family of small heat-shock proteins (sHsps). According to the present model, based on the in vitro experiments, sHsps are molecular chaperones that bind and prevent aggregation of nonnative proteins during heat shock. Previously, we have shown that IbpA and IbpB bind to endogenous E. coli proteins aggregated intracellularly by heat shock, which can be separated from soluble proteins and membranes in sucrose density gradients (fraction S). In this work we have found that marine bacterium Vibrio harveyi contains a single sHsp which is strongly induced by heat shock and reacts with the anti-IbpA/B serum. The 26 amino-terminal amino acids of this sHsp bear high homology to E. coli IbpA and IbpB proteins (73% and 54% identity, respectively). Fraction S was prepared from heat-shocked cells of V. harveyi, it contained high amounts of the IbpA/B protein. This result indicates that the IbpA/B protein of V. harveyi binds to the proteins that aggregate in V. harveyi cells during heat shock. Received October 15, 2000; accepted January 30, 2001.     

Importance of N- and C-terminal regions of IbpA, Escherichia coli small heat shock protein, for chaperone function and oligomerization

Small heat shock proteins are ubiquitous molecular chaperones that, during cellular stress, bind to misfolded proteins and maintain them in a refolding competent state. Two members of the small heat shock protein family, IbpA and IbpB, are present in Escherichia coli. Despite 48% sequence identity, the proteins have distinct activities in promoting protein disaggregation. Cooperation between IbpA and IbpB is crucial for prevention of the irreversible aggregation of proteins. In this study, we investigated the importance of the N- and C-terminal regions of IbpA for self-oligomerization and chaperone functions. Deletion of either the N- or C-terminal region of IbpA resulted in a defect in the IbpA fibril formation process. The deletions also impaired IbpA chaperone function, defined as the ability to stabilize, in cooperation with IbpB, protein aggregates in a disaggregation-competent state. Our results show that the defect in chaperone function, observed in truncated versions of IbpA, is due to the inability of these proteins to interact with substrate proteins and consequently to change the properties of aggregates. At the same time, these versions of IbpA interact with IbpB similarly to the wild type protein. Competition experiments performed with the pC peptide, which corresponds to the IbpA C terminus, suggested the importance of IbpA intermolecular interactions in the stabilization of aggregates in a state competent for disaggregation. Our results suggest that these interactions are not only dependent on the universally conserved IEI motif but also on arginine 133 neighboring the IEI motif. IbpA mutated at arginine 133 to alanine lacked chaperone activity.     

A model for heterooligomer formation in the heat shock response of Escherichia coli

Small heat shock proteins (sHsp) are widely distributed molecular chaperones that bind to misfolded proteins to prevent irreversible aggregation and aid in refolding to a competent state. The sHsps characterized thus far all contain a conserved α-crystallin, and variable N- and C-termini critical for chaperone activity and oligomerization. The Escherichia coli sHsps IbpA and IbpB share 48% sequence homology, are induced by heat shock and oxidative stress, and each requires the presence of the other to effect protein protection. Molecular Dynamics (MD) simulations of homology-modeled monomers and heterooligomers of these sHsps identify a possible mechanism for cooperation between IbpA and IbpB.     

Effects of the IbpAB AND ClpA chaperones on DnaKJE-dependent refolding of bacterial luciferases in Escherichia coli cells

The rate and level of DnaKJE-dependent refolding of the thermoinactivated Aliivibrio fischeri luciferase are considerably lower in Escherichia coli ibpA and ibpB mutants than in wild type cells. The rate and level of refolding are lower in E. coli ibpB::kan than in ibpA::kan cells. The decline of refoldings level in E. coli clpA::kan makes progress only with the increase of thermoinactivation time of luciferase. Plasmids with the genes ibpAB don't compensate clpA mutation. It is supposed that small chaperones IbpAB and chaperone ClpA operate independently in a process of DnaKJE-dependent refolding of proteins at the different stages.     

Trimethoprim induces heat shock proteins and protein aggregation in E. coli cells

Trimethoprim (TMP), an inhibitor of dihydrofolate reductase, decreases the level of tetrahydrofolate supplying one-carbon units for biosynthesis of nucleotides, proteins, and panthotenate. We have demonstrated for the first time that one of the effects of the TMP action in E. coli cells is protein aggregation and induction of heat shock proteins (Hsps). TMP caused induction of DnaK, DnaJ, GroEL, ClpB, and IbpA/B Hsps. Among these Hsps, IbpA/B were most efficiently induced by TMP and coaggregated with the insoluble proteins. Upon folate stress, deletion of the delta ibpA/B operon resulted in increased protein aggregation but did not influence cell viability.     

Differential degradation for small heat shock proteins IbpA and IbpB is synchronized in Escherichia coli: Implications for their functional cooperation in substrate refolding

Small heat shock proteins (sHSPs), as a conserved family of ATP-independent molecular chaperones, are known to bind non-native substrate proteins and facilitate the substrate refolding in cooperation with ATP-dependent chaperones (e.g., DnaK and ClpB). However, how different sHSPs function in coordination is poorly understood. Here we report that IbpA and IbpB, the two sHSPs of Escherichia coli, are coordinated by synchronizing their differential in vivo degradation. Whereas the individually expressed IbpA and IbpB are respectively degraded slowly and rapidly in cells cultured under both heat shock and normal conditions, their simultaneous expression leads to a synchronized degradation at a moderate rate. Apparently, such synchronization is linked to their hetero-oligomerization and cooperation in binding substrate proteins. In addition, truncation of the flexible N- and C-terminal tails dramatically suppresses the IbpB degradation, and somehow accelerates the IbpA degradation. In view of these in vivo data, we propose that the synchronized degradation for IbpA and IbpB are crucial for their synergistic promoting effect on DnaK/ClpB-mediated substrate refolding, conceivably via the formation of IbpA–IbpB-substrate complexes. This scenario may be common for different sHSPs that interact with each other in cells.     

Overexpression of IbpB enhances production of soluble active Streptomyces olivaceovirdis XynB in Escherichia coli

The small heat shock protein IbpB of Escherichia coli can accelerate protein disaggregation from inclusion body by Hsp100-Hsp70 re-activation system in vitro. It was therefore hypothesized that overexpression of IbpB might be able to promote protein disaggregation from inclusion body, by which more soluble recombinant proteins would be obtained. The overexpression of IbpB actually enhanced production of more active soluble XynB of Streptomyces olivaceovirdis in E. coli BL21(DE3). Surprisingly, the disaggregation of XynB from inclusion body was not accelerated. It seemed that the overexpressed IbpB protected improperly or partially folded XynB from aggregation and mediated the subsequent refolding. These results show potential of improving production of active heterologous proteins in E. coli.     

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co-operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co-operatively the DnaK system in reversing protein aggregation. A delta ibpAB delta clpB double mutant exhibits strongly increased protein aggregation at 42 degrees C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37 degrees C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for delta ibpAB, delta clpB, and the double delta ibpAB delta clpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.