Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli

The kinetics of the heat-shock response and the formation of inclusion bodies in recombinant Escherichia coli TG1 were studied in glucose-limited high-cell-density cultures in response to temperature-induced production of human basic fibroblast growth factor (hFGF-2), a protein which partially aggregates into inclusion bodies. The maximum synthesis rates of heat-shock proteins were similar to those in a control cultivation with a strain carrying an expression vector without inducible structural gene. However, the maximum of induction for many heat-shock proteins including DnaK, ClpB, and HtpG was reached at least 30 min later when synthesis of hFGF-2 was simultaneously induced by the temperature upshift. During this first production phase, hFGF-2 was exclusively deposited in the insoluble cell fraction. Thereafter, accumulation of soluble hFGF-2 was observed, too, indicating that the recombinant protein needs heat-shock chaperones for proper folding at elevated temperatures. Strong recombinant protein production prolonged the synthesis of the majority of heat-shock proteins (including GroELS, DnaK, ClpB, and HtpG) even in a wildtype dnaK(+) background. In contrast, the synthesis rates of the small heat-shock proteins IbpA and IbpB declined within 1 h to preinduction values in control and hFGF-2 producing cultures. In the producing cultivation, IbpA and IbpB synthesis ceased to an undetectable level when soluble hFGF-2 started to accumulate, whereas the synthesis rates of the other heat-shock proteins including those belonging to the DnaK and GroEL families remained high throughout the entire production phase.     

A cya deletion mutant of Escherichia coli develops thermotolerance but does not exhibit a heat-shock response

An adenyl cyclase deletion mutant (cya) of E. coli failed to exhibit a heat-shock response even after 30 min at 42 degrees C. Under these conditions, heat-shock protein synthesis was induced by 10 min in the wild-type strain. These results suggest that synthesis of heat-shock proteins in E. coli requires the cya gene. This hypothesis is supported by the finding that a presumptive cyclic AMP receptor protein (CRP) binding site exists within the promoter region of the E. coli htpR gene. In spite of the absence of heat-shock protein synthesis, when treated at 50 degrees C, the cya mutant is relatively more heat resistant than wild type. Furthermore, when heat shocked at 42 degrees C prior to exposure at 50 degrees C, the cya mutant developed thermotolerance. These results suggest that heat-shock protein synthesis is not essential for development of thermotolerance in E. coli.     

Response dynamics of 26-, 34-, 39-, 54-, and 80-kDa proteases in induced cultures of recombinant Escherichia coli

Several researchers have demonstrated that the presence of a heterologous protein in recombinant Escherichia coli elicits a response similar to the heat-shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity in E. coli that are "shocked" by the induction of a recombinant protein, CAT, which is an endogenous protein in some E. coli strains. A novel, sodium dodecyl sulfate gelatin poly-acrylamide gel electrophoresis (SDS-GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent-like stress response. (c) 1993 John Wiley & Sons, Inc.     

Fatty acid profile of Escherichia coli during the heat-shock response.

The possible changes in the fatty acid profile of Escharichia coli during heat-shock have been investigated. Bacteria growing in steady-state at 30 degrees C were subjected to an abrupt temperature upshift to 45 degrees C and held at the high temperature for various periods of time in order to elicit the heat-shock response. Fatty acid compositions of lipids extracted from samples taken at different times after the temperature upshift, as well as from cultures in steady-state at 30 and 45 degrees C, were determined by gas-chromatography. It has been found that the total unsaturates to total saturates ratio decreases gradually during heat-shock and that 30 min after the temperature jump, the reduction is equivalent to 57% of the difference between ratios corresponding to steady-state cultures at 30 and 45 degrees C. Consistent with this remodeling of lipid acyl chains, there is a decrease in the excimerization rate of the fluidity probe dipyrenylpropane incorporated into sonicated E. coli lipid extracts. Such modifications occur within the time-span of the heat-shock response, as judged from our previous measurements of the kinetics of change in heat-shock proteins induction ratio. Together, these results indicate that the control of membrane fluidity during the heat-shock response can be accounted for, at least in part, by an important change in the fatty acid composition of Escherichia coli lipids.     

Structured model to predict intracellular amino acid shortages during recombinant protein overexpression in E. coli.

Recombinant protein overexpression and the classical stringent response have been shown to induce the same proteases. Since the stringent response was the result of an intracellular amino acid shortage, it was hypothesized that the overexpression of the recombinant protein also caused an intracellular amino acid shortage. A structured non-segregated kinetic mathematical model of recombinant Escherichia coli was developed to predict intracellular amino acid shortages during recombinant protein overexpression, and thus the induction of the stringent response. Two model recombinant proteins were examined, chloramphenicol acetyl-transferase (CAT) and an 'average protein'. The model predicted an aromatic amino acid shortage during CAT overexpression, as predicted based on the CAT's amino acid content. The model also predicted a shortage of the intracellular alanine family amino acid pool during CAT overexpression. This was unexpected due to the relatively low content of alanine family amino acids in CAT compared to the average E. coli protein. The model predicted alanine, glutarate, and aspartate family amino acid shortages during recombinant 'average protein' overexpression. Additionally, the model predicted a decrease in the ribosome pool at induction for both recombinant proteins, which agrees with published experimental results. Thus, the structured kinetic model was able to predict amino acid shortages, that could potentially cause a stringent response and elevated protease activity.     

On-line estimation of the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins by monitoring respiratory energy generation.

Human basic fibroblast growth factor (hFGF-2) was produced in high-cell density cultures of recombinant Escherichia coli using a temperature-inducible expression system. The synthesis rates of proteins were followed by two-dimensional gel electrophoresis of the (35)S-methionine-labeled proteom. After temperature induction of hFGF-2 synthesis, the rate of total protein synthesis per biomass increased by a factor of three, mainly as a result of the additional synthesis of hFGF-2 and heat-shock proteins. The synthesis rates of heat-shock proteins and constitutive plasmid-encoded proteins increased after the temperature upshift also in the control strain without hFGF-2 gene but followed time profiles different from the producing strain. The energy demand for the extra synthesis of plasmid-encoded and heat-shock proteins resulted in an elevated respiratory activity and, consequently, in a reduction of the growth rate and the biomass yield. A procedure was developed to relate the energy demand for the additional synthesis of these proteins to the generation of energy in the respiratory pathway. Specific energy production was estimated based on on-line measurable rates of oxygen consumption, or carbondioxide evolution and growth, respectively. In this way, the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins was quantified from on-line accessible data.     

Localized heat-shock induction in Drosophila melanogaster

We describe a technique for inducing localized expression of genes fused to heat-shock gene promoters. We demonstrate that a localized heat-shock response can be induced in Drosophila melanogaster at any developmental stage after formation of the cellular blastoderm by contacting a region of the animal with a heated needle. The size of the induced region can be altered by varying parameters such as the temperature and size of the needle tip. The test system utilized here is a D. melanogaster strain transformed with a fusion of the Drosophila hsp26 gene and the E. coli lacZ gene; the activity of this hybrid gene is monitored in whole animals by staining for beta-galactosidase activity. Induced beta-galactosidase activity is confined to the cells in the region of heating; the beta-galactosidase activity can still be detected 48 hr after the heat shock. Given the heat inducibility of Drosophila heat-shock promoters in heterologous systems, we suggest that this technique will be useful for allowing spatially controlled induction of a gene of interest in any organism into which fusion genes can be introduced. Additional uses of the technique for following cell movements during development are discussed.     

Developmental control of the heat-shock stress regulon in Streptomyces coelicolor

In the differentiating eubacterium Streptomyces coelicolor , nutritional imbalances activate a developmental programme which involves the heat-shock stress regulon. In liquid batch cultures, the growth curve could be separated into four components: rapid growth 1 (RG1), transition (T), rapid growth 2 (RG2) and stationary (S). Patterns of gene expression in cultures subjected to heat shock in various phases were recorded on two-dimensional gels and analysed using advanced statistical methods. The responses of all heat-shock proteins (HSPs) were highly dependent upon the growth phase, thus demonstrating that the four phases of growth were physiologically distinct. For many HSPs, the levels of thermal induction attained were closely related to growth stage-determined levels of synthesis before heat shock, thus supporting the idea that developmental and thermal induction of this stress regulon have common control elements. Cluster analysis identified five groups of HSPs displaying similar kinetics of heat and developmentally induced synthesis, probably reflecting the influence of major regulatory systems. Methods introduced here to analyse the response of groups of genes to multiple simultaneous stimuli should find broad applications to studies of other prokaryotic and eukaryotic regulons.     

Acidic extracellular environment induces only a subset of heat-shock proteins in primary mouse kidney cell cultures

Exposure of primary mouse kidney cell cultures to acidic medium (pH 5.5) induced the expression of a 70 kilodalton (kDa) protein. This protein was identified as the major inducible heat-shock protein 70 (hsp70) by immunoprecipitation with anti-hsp70 serum and Northern blot analysis with a hsp70 cDNA probe. Maximum induction of the 70-kDa protein at pH 5.5 after 240 min was about 30% of that observed after 60 min of thermal treatment at 43 degrees C. In addition, there was an apparent induction of the glucose-regulated proteins (GRPs) of 76-78 and 98-100 kDa, but not of the other hsps. This subset induction of the heat-shock response by acidic medium suggests that different mechanisms are responsible for the induction of the various families of hsps.     

The major heat-shock protein hsp 68 is not induced by stress in mouse erythroleukemia cell lines

Most mammalian cells respond to brief incubation at elevated temperatures by enhanced or new synthesis of a set of heat-shock proteins (hsp). In mouse cells, as determined by SDS--one-dimensional gel electrophoresis, the most prominent hsps have molecular masses of approximately 89,000, 70,000, and 68,000 Da. When the heat-shock response of the mouse erythroleukemia cell line D1B, or two other DBA/2 cell lines (707C1 and 745C2), was examined by [35S]methionine labelling, following heat shocks of 10 min at 42 or 44 degrees C, or 1 h at 45 degrees C, no protein band corresponding to hsp 68 was observed. However, the synthesis of both hsp 89 and hsp 70 was enhanced. Northern blot analysis of cytoplasmic RNA extracted from control and stressed cells indicated that hsp 68 mRNA was absent, even after stresses of up to 1 h at 45 degrees C. Differentiation induced by dimethyl sulphoxide (DMSO) (monitored by the induction of globin synthesis) had no effect on hsp 68 expression in D1B cells; also, hsp 68 could not be induced at various stages of differentiation (0-72 h). Southern blot analysis showed that all three hsp-68 genes were present and not rearranged, and apparently did not carry any deletion in their 5' ends. To determine whether methylation could be involved in maintaining the genes in their silent state, we treated cells with 10 microM 5-azacytidine for 48 h. No hsp 68 expression was observed following such treatment in either undifferentiated or DMSO-induced differentiated D1B cells. Furthermore, Southern blot analysis of MspI/HpaII-digested genomic D1B DNA did not display any differences in methylation patterns around the promoter region of the probed gene compared with control cells, indicating that methylation is not involved in hsp-68 repression. When chimeric plasmids carrying the bacterial chloramphenicol acetyl transferase gene under regulation of the mouse hsp-68 or Drosophila hsp-70 promoters were transfected into D1B cells, minimal (2-fold) or no induction was observed, in contrast with the 60-fold induction seen in a control myeloma cell line. These results suggest a trans-acting mechanism of hsp-68 repression in erythroleukemia cells.     

Control of growth and recombinant protein synthesis by heat-shock in a mutant mammalian cell line

Summary The effect of heat-shock on cell growth and the induction of recombinant protein synthesis by a temperature-sensitive (ts) Chinese hamster ovary (CHO) cell line was investigated. An optimal regime of successive 2 hour heat-shocks (42°C) over 72 hours was found to simultaneously arrest cell growth and induce the synthesis of recombinant protein. The enhanced induction achieved from growth arrested cells may find application in the production of cytotoxic proteins.     

Effect of heat shock on expression of proteins not involved in the heat-shock regulon.

The effect of heat shock on the expression of some genes of Escherichia coli was tested. To avoid side effects, promoters of the genes were fused to lacZ and their expression measured by the level of beta-galactosidase. The results show that expression of umuC, recA and polB, after induction of the SOS response, was somewhat higher in the heat-shocked than in the non-shocked cells, whereas expression of ada, alkB and alkA genes, after induction of the adaptive response, was about the same. Unexpectedly, it was found that expression of lacZ from its own promoter was drastically lowered in the heat-shocked cells. This effect, however, seems not to be dependent on the induction of heat-shock proteins.     

Effect of heat-shock proteins for relieving physiological stress and enhancing the production of penicillin acylase in Escherichia coli

High-level expression of recombinant penicillin acylase (PAC) using the strong trc promoter system in Escherichia coli is frequently limited by the processing and folding of PAC precursors (proPAC) in the periplasm, resulting in physiological stress and inclusion body formation in this compartment. Periplasmic heat-shock proteins with protease or chaperone activity potentially offer a promise for overcoming this technical hurdle. In this study, the effect of the two genes encoding periplasmic heat-shock proteins, that is degP and fkpA, on pac overexpression was investigated and manipulation of the two genes to enhance the production of recombinant PAC was demonstrated. Both DeltadegP and DeltafkpA mutants showed defective culture performance primarily due to growth arrest. However, pac expression level was not seriously affected by the mutations, indicating that the two proteins were not directly involved in the pathway for periplasmic processing of proPAC. The growth defect caused by the two mutations (i.e., DeltadegP and DeltafkpA) was complemented by either one of the wild-type proteins, implying that the function of the two proteins could partially overlap in cells overexpressing pac. The possible role that the two heat-shock proteins played for suppression of physiological stress caused by pac overexpression is discussed.