Probing the Role of Protein Folding in Inclusion Body Formation

Chrunyk, Boris A.; Evans, Judy; Wetzel, Ronald

doi:10.1021/bk-1993-0526.ch004

Cited by 13 publications

(23 citation statements)

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“…We interpreted this data to suggest that differences in the thermodynamic stability of the native state cannot account for mutational effects on IB formation in this series of mutants [6]. Other ways in which mutational and thermal effects might be mediated are: (a) effects on the solubility or kinetic lifetime of a transient folding intermediate; or (b) effects on a chaperone interaction or some other component of the biosynthetic machinery.…”

Section: Resultsmentioning

confidence: 92%

“…Both WT and the K97V of IL-lb were purified from the supernatant of a native lysate of E. coli as described previously [6]. For refolding studies.…”

Section: Aggregation Studiesmentioning

confidence: 99%

“…Although exhibiting higher thermodynamic stability than wild-type IL-l/3, a much larger percentage of total IL-18 (K97V) is deposited into IBs than is the wild-type molecule [6]. Like a number of other IB-forming proteins [4,11], the proportion of the K97V mutant deposited into IBs significantly decreases when growth temperature is decreased [6]. We report here excellent correspondence between these temperature effects on IB formation by sequence variants and the temperature effects on aggregation in vitro when the same proteins are refolded from denaturant.…”

Section: Introductionmentioning

confidence: 99%

“…Recently we described a series of point mutants in the small, monomeric B-barrel protein interleukin-lb (IL-1B) which exhibit wide variation in the extent to which they are deposited into IBs when expressed in E. coli [6].…”

Section: Introductionmentioning

confidence: 99%

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Inclusion body formation by interleukin‐1β depends on the thermal sensitivity of a folding intermediate

Wetzel

Chrunyk

1994

FEBS Letters

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We show that sequence and growth temperature effects on IB formation in the small, monomeric B-barrel protein interleukin-lp (IL-lb) can be quantitatively reproduced in an in vitro system in which IL-l/l is refolded from denaturant at different temperatures.The results suggest that temperature and mutational effects on IB formation may be based on intrinsic properties of the protein sequence rather than interactions with chaperones or other cellular factors. We also report striking correlations of IB formation with mutation-dependent changes in residue hydrophobicity.The nature of these trends differs considerably with residue position, however, suggesting that they are mediated by particular local environments created by an ordered structure.

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Section: Resultsmentioning

confidence: 92%

“…Both WT and the K97V of IL-lb were purified from the supernatant of a native lysate of E. coli as described previously [6]. For refolding studies.…”

Section: Aggregation Studiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Inclusion body formation by interleukin‐1β depends on the thermal sensitivity of a folding intermediate

Wetzel

Chrunyk

1994

FEBS Letters

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“…During the folding of P22 tailspike proteins with tsf (temperature-sensitive folding) amino acid substitutions, a folding intermediate is aggregation-prone at high temperatures (16 -19). Other proteins such as interluekin-1␤ (20) and bovine growth hormone (21) have similar tendencies to aggregate.…”

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confidence: 99%

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Aramli

Teschke

2001

Journal of Biological Chemistry

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Understanding the nature of protein grammar is critical because amino acid substitutions in some proteins cause misfolding and aggregation of the mutant protein resulting in a disease state. Amino acid substitutions in phage P22 coat protein, known as tsf (temperature-sensitive folding) mutations, cause folding defects that result in aggregation at high temperatures. We have isolated global su (suppressor) amino acid substitutions that alleviate the tsf phenotype in coat protein (Aramli, L. A., and Teschke, C. M. (1999) J. Biol. Chem. 274, 22217-22224). Unexpectedly, we found that a global su amino acid substitution in tsf coat proteins made aggregation worse and that the tsf phenotype was suppressed by increasing the rate of subunit assembly, thereby decreasing the concentration of aggregation-prone folding intermediates.The primary amino acid sequence of a polypeptide encodes all of the information necessary for folding and assembly pathways, as well as the native three-dimensional structure (2). Substitutions and deletions in the amino acid sequence of a protein can have a significant impact on the ability of a protein to fold or assemble properly. Depending on the protein, such changes in the amino acid sequence can lead to protein misfolding, mislocalization caused by misfolding, or aggregation (3, 4). Amino acid substitutions in p53 lead to a misfolding problem compromising the function of the protein, resulting in cancer (5). Further, there are the diseases that result from mislocalization caused by protein misfolding. For example, in ␣ 1 -antitrypsin deficiency, a single amino acid substitution results in the misfolding of ␣ 1 -antitrypsin, leading to the accumulation of long chain polymers within the hepatocyte. This leads to a reduction of the plasma concentrations of ␣-antitrypsin and predisposes individuals to emphysema and liver disease (6). Therefore, understanding the nature of protein grammar is of paramount interest.Although the process of protein folding is still not completely understood, it is known that larger proteins often have identifiable folding intermediates. These folding intermediates may interact inappropriately before reaching the native state. In fact, protein misfolding is a common problem faced by biotechnology companies that harvest proteins of commercial interest using recombinant DNA technologies in heterologous hosts (7-11). Often these proteins have problems with inclusion body formation, thereby decreasing the yield of pharmaceutically important products. Single amino acid substitutions can affect the folding pathway by shifting the folding from the productive pathway to off-pathway aggregation. For example, the amino acid substitutions in transthyretin causes a shift in the equilibrium between the native state and an aggregation-prone unfolding intermediate, resulting in amyloid formation. Individuals with any of the 50 known amino acid substitutions in transthyretin are predisposed to familial amyloidosis (12-15). During the folding of P22 tailspike proteins with tsf (te...

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Recombinant Human Retinol-Binding Protein Refolding, Native Disulfide Formation, and Characterization

Xie

Lashuel

Miroy

et al. 1998

Protein Expression and Purification

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Probing the Role of Protein Folding in Inclusion Body Formation

Cited by 13 publications

References 0 publications

Inclusion body formation by interleukin‐1β depends on the thermal sensitivity of a folding intermediate

Inclusion body formation by interleukin‐1β depends on the thermal sensitivity of a folding intermediate

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Recombinant Human Retinol-Binding Protein Refolding, Native Disulfide Formation, and Characterization

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