Kinetics of inclusion body formation studied in intact cells by FT‐IR spectroscopy

Ami, Diletta; Natalello, Antonino; Gatti‐Lafranconi, Pietro; Lotti, Marina; Doglia, Silvia Maria

doi:10.1016/j.febslet.2005.04.085

Cited by 92 publications

(75 citation statements)

References 15 publications

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“…The conformational background sustaining the IB molecular structure lies on an extended, intermolecular ␤-sheet architecture (6) that coexists with various amounts of a population of native-like, correctly folded polypeptides (1)(2)(3)26). Such a ␤-sheet pattern is progressively lost at the expense of native-like structure when the temperature at which IBs are formed decreases (19,28), indicating the existence of several categories of protein aggregates in regard to their molecular organization and even global morphology (13).…”

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confidence: 99%

Localization of Functional Polypeptides in Bacterial Inclusion Bodies

García‐Fruitós

Arı́s

Villaverde

2007

Appl Environ Microbiol

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Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a ␤-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular ␤-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.Procedures for in vitro protein refolding are under continuous development (22,32,35), since many proteins of industrial or pharmacological interest are produced in recombinant microorganisms, especially bacteria, as insoluble aggregates called inclusion bodies (IBs) (38). Recent insights into the structure and physiology of bacterial IBs have revealed that at least a significant fraction of the embedded protein occurs in a properly folded native-like form (36) and that for aggregates formed by enzymes, this fact is reflected by the occurrence of enzymatic activity associated with these particles (16,34,39). While for hormones or other drugs to be used in vivo, in vitro solubilization of IBs and refolding of IB proteins would still be required to allow their proper use (27), enzymes to be used in bioprocesses could be employed straight after production, skipping any refolding step. This is particularly appealing since the specific activity found in IB enzymes, although variable when comparing different protein species, is not dramatically different from that exhibited by the soluble counterparts (15, 16), and on the other hand, refolding procedures render yields of active protein that are usually far from 100%.Apart from the obvious potential of enzyme IBs as catalyzers, the occurrence of properly folded, active enzymes poses intriguing structural questions. The conformational background sustaining the IB molecular structure l...

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confidence: 99%

Localization of Functional Polypeptides in Bacterial Inclusion Bodies

García‐Fruitós

Arı́s

Villaverde

2007

Appl Environ Microbiol

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“…This work highlights the potential of FT-IR spectroscopy not only to study protein aggregation in IBs [6][7][8], but also to monitor the membrane response induced by the heterologous production.…”

Section: Resultsmentioning

confidence: 92%

FT-IR spectroscopy for the study of bacterial membrane stress induced by recombinant protein production

et al. 2006

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BackgroundMicroorganisms respond to environmental stresses regulating their membrane fluidity by changing the lipid fatty acid composition. In particular, in bacterial cells the alteration of membrane lipid composition plays an important role in response to heat and toxic stresses [1,2]. It is known that at increasing temperatures the regulation of membrane fluidity occurs through the incorporation of more saturated fatty acids, longer acyl chains and, in some cases, by changing the unsaturated fatty acids from cis to trans conformation. Among different stresses, production of heterologous proteins is of particular interest for its applications in biotechnology. ResultsIn this work we show the potential of FT-IR microspectroscopy to monitor changes in membrane composition during recombinant protein production. In particular, we studied the infrared absorption of E. coli strains expressing several proteins with different amount of soluble and aggregated fractions. In addition, as a control experiment, we have studied E. coli strains expressing beta galactosidase, as reporter gene, under the chaperone IbpB promoter [3].In all model systems examined, an increase in the band intensities at 2850 cm -1 and at 2925 cm -1 was observed in presence of a high level of stress [4]. As these two bands are due to CH 2 stretching vibrations [5], this spectral behaviour may indicate that longer acyl chain and /or more saturated fatty acids are incorporated in cell membranes. The protein expression analysis indicates that the enzymes that lead to the accumulation of Acetyl Co-A (precursor of fatty acids) are strongly accumulated whereas the phospholipide degradation seems being inhibited.Indeed, when the protein is expressed also in a soluble form -as indicated by the FT-IR protein response in the amide I region and confirmed by the SDS-PAGE analysis -the two CH 2 bands are more intense than in the case of IB formation. This behaviour seems to indicate that the presence of the soluble recombinant protein induces a stress in the cell, suggesting that IBs could limit the toxicity of overexpressed foreign proteins. ConclusionThis work highlights the potential of FT-IR spectroscopy not only to study protein aggregation in IBs [6][7][8], but also to monitor the membrane response induced by the heterologous production.

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“…The method enables us to monitor the kinetics of aggregate formation within intact cells in a rapid and non invasive way and to obtain structural information on proteins within IBs. We will report results on four recombinant proteins: human growth hormone (h-GH), human interferon-alpha-2b (IFN-alpha-2b) [4,7], Pseudomonas fragi lipase [3], and green fluorescent protein -glutathione S-transferase fusion protein (GFP-GST) [8].…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, it has been suggested that the presence of native-like structures within IBs [1][2][3][4] improves the efficiency of refolding protocols that employ mild solubilization methods [5]. This property could also explain the residual enzymatic activity of recombinant proteins in IBs, with possible applications in biocatalysis [6].…”

Section: Introductionmentioning

confidence: 99%