FT-IR study of heterologous protein expression in recombinant Escherichia coli strains

Ami, Diletta; Bonecchi, Loredana; Calı̀, Simona; Orsini, Gaetano; Tonon, Giancarlo; Doglia, Silvia Maria

doi:10.1016/j.bbagen.2003.09.008

Cited by 54 publications

(48 citation statements)

References 13 publications

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“…It is interesting to point out that, when the recombinant protein is expressed at high levels as IBs, the FT-IR detection of IB formation can be easily done by evaluating the aggregate band intensity directly in the second derivative spectrum of the producer cells [7]. In this case, the intense aggregate band is not hidden by the overall absorption of the total cell protein content, not requiring the subtraction of the control cell spectrum.…”

Section: Kinetics Of Inclusion Body Formation Under Differentmentioning

confidence: 99%

“…Indeed, the second derivative analysis of the protein Amide I band [10][11][12] allows determining the protein secondary structure, since Amide I is a structure sensitive band, being mainly due to the absorption of the carbonyl C‚O group of the peptide bond. Interestingly, it has been shown that the analysis of the Amide I band components enables also the detection of protein aggregates [4,6,7,13], which are not easily monitored by other optical spectroscopies.…”

Section: Detection Of Inclusion Bodies In Vivo By Ft-ir Spectroscopymentioning

confidence: 99%

“…To eliminate the contribution of the total cell proteins not encoded by the lipase gene, the control cell spectrum was then subtracted from that of the producer cells (Fig. 1B) [7]. This difference spectrum represents the spectral response of the heterologous protein within the cells that can be better appreciated in the second derivative spectrum (Fig.…”

Section: Detection Of Inclusion Bodies In Vivo By Ft-ir Spectroscopymentioning

confidence: 99%

“…The presence of aggregates was investigated in intact E. coli cells expressing the heterologous lipase by the FT-IR microspectroscopy approach recently developed [7].…”

Section: Detection Of Inclusion Bodies In Vivo By Ft-ir Spectroscopymentioning

confidence: 99%

“…In the last decade, FT-IR spectroscopy did emerge as a powerful tool to explore different aspects of the process of protein aggregation [3][4][5][6]. More recently, the study of aggregated recombinant proteins within intact cells has been made possible by the use of FT-IR microspectroscopy on small amounts of samples from fermentation broths [7]. In this paper, we show how this approach is suitable to monitor the kinetics of IB formation -in real time within intact cellsby following the expression in Escherichia coli of a recombinant lipase from Pseudomonas fragi (PFL) at two different temperatures of expression.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2005

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The aggregation of a recombinant lipase as inclusion bodies (IBs) was studied directly within intact Escherichia coli cells by FT-IR microspectroscopy. Through this approach, it was possible to monitor in real time the different kinetics of IB formation at 37 and 27°C, in excellent agreement with the results of the SDS-PAGE analysis. Furthermore, insights on the residual native-like structure of the expressed protein within IB -both isolated and inside cells -were obtained by the secondary structure analysis of the Amide I band in the IB FT-IR spectra.

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Section: Kinetics Of Inclusion Body Formation Under Differentmentioning

confidence: 99%

Section: Detection Of Inclusion Bodies In Vivo By Ft-ir Spectroscopymentioning

confidence: 99%

Section: Detection Of Inclusion Bodies In Vivo By Ft-ir Spectroscopymentioning

confidence: 99%

“…The presence of aggregates was investigated in intact E. coli cells expressing the heterologous lipase by the FT-IR microspectroscopy approach recently developed [7].…”

Section: Detection Of Inclusion Bodies In Vivo By Ft-ir Spectroscopymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2005

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Structural Properties of Bacterial Inclusion Bodies

Natalello¹,

Ami²,

Doglia³

2014

Protein Aggregation in Bacteria

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Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

Doglia

Ami

Natalello

et al. 2008

Biotechnology Journal

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The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co-expression of chaperones.

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FT-IR study of heterologous protein expression in recombinant Escherichia coli strains

Cited by 54 publications

References 13 publications

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

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

Structural Properties of Bacterial Inclusion Bodies

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

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