Does DsbA Have Chaperone-like Activity?

Zheng, Weidong; Quan, Hui; Song, Jiu-li; Yang, Shengli; Wang, Chih-chen

doi:10.1006/abbi.1996.9783

Cited by 43 publications

(41 citation statements)

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“…The specific TPOR activity of DsbC is 28 Ϯ 4% that of PDI (169 mol GSSG/min/g). It is to be noted that DsbC has a much higher isomerase activity than that of DsbA, which shows only about 5% that of PDI (23). Fig.…”

Section: Resultsmentioning

confidence: 88%

“…However, compared with eukaryotic PDI, the in vitro isomerase activity is about 30% for DsbC and only 5% for DsbA (23).…”

Section: Discussionmentioning

confidence: 90%

“…The high-resolution crystal structure analysis of DsbA (22) has shown that an extensive uncharged surface surrounding the active-site disulfide is responsible for peptide binding. We have also presented evidence for the chaperone activity of DsbA in assisting the in vitro refolding of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (23). Recently, Darby et al (24) reported more substantial noncovalent interactions between peptides with DsbC than with DsbA.…”

mentioning

confidence: 72%

“…S-Carboxymethylated A chain of insulin was prepared as described previously (23). Two peptides, YTRDLVYKNP (10-mer peptide) and (YSPTSPS) 2 (14-mer peptide), were synthesized by solid phase method using t-butoxycarbonyl strategy and purified with Sephadex G-10 and reverse phase-high performance liquid chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…As GAPDH has no disulfide bond, the abilities of DsbC to increase reactivation of the denatured enzyme with concurrent suppression of aggregation during refolding can only be ascribed to its chaperone activity independent from its isomerase activity, as in the cases for PDI (11,13) and DsbA (23).…”

Section: Effects Of Dsbc On the Reactivation And Aggregation Of Gdnhcmentioning

confidence: 99%

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Chaperone Activity of DsbC

Chen¹,

Song²,

Zhang³

et al. 1999

Journal of Biological Chemistry

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163

107

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DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys 98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10-and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.

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Section: Resultsmentioning

confidence: 88%

“…However, compared with eukaryotic PDI, the in vitro isomerase activity is about 30% for DsbC and only 5% for DsbA (23).…”

Section: Discussionmentioning

confidence: 90%

mentioning

confidence: 72%

Section: Methodsmentioning

confidence: 99%

Section: Effects Of Dsbc On the Reactivation And Aggregation Of Gdnhcmentioning

confidence: 99%

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Chaperone Activity of DsbC

Chen¹,

Song²,

Zhang³

et al. 1999

Journal of Biological Chemistry

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163

107

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Secretory production of human leptin inEscherichia coli

Jeong

Lee

2000

Biotechnol. Bioeng.

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Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis. In this study, human leptin was produced and secreted efficiently in Escherichia coli using a novel Bacillus sp. endoxylanase signal peptide. The endoxylanase signal sequence consisted of 28 amino acids (84 bp) was fused to the leptin structural gene. The fused gene was expressed using an inducible promoter (T7 or Trc) by adding 1 mM IPTG. Using T7 promoter in E. coli BL21(DE3), most of protein produced was in a premature form. Using the Trc promoter, which is weaker than T7, leptin was efficiently produced and secreted as a mature form (40% of total proteins) at 37°C. However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E. coli. At 30°C, ca. 90% of leptin was produced in a soluble form, but the total amount of leptin produced was 40% less than that obtained at 37°C. When the periplasmic oxidoreductase of E. coli, DsbA, was co‐expressed, 69% of the secreted leptin (26% of total proteins) was produced as soluble form at 37°C without the decrease of the amount of leptin produced. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 398–407, 2000.

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Secretory production of human leptin in Escherichia coli

Jeong

Lee

2000

Biotechnol. Bioeng.

View full text Add to dashboard Cite

Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis. In this study, human leptin was produced and secreted efficiently in Escherichia coli using a novel Bacillus sp. endoxylanase signal peptide. The endoxylanase signal sequence consisted of 28 amino acids (84 bp) was fused to the leptin structural gene. The fused gene was expressed using an inducible promoter (T7 or Trc) by adding 1 mM IPTG. Using T7 promoter in E. coli BL21(DE3), most of protein produced was in a premature form. Using the Trc promoter, which is weaker than T7, leptin was efficiently produced and secreted as a mature form (40% of total proteins) at 37 degrees C. However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E. coli. At 30 degrees C, ca. 90% of leptin was produced in a soluble form, but the total amount of leptin produced was 40% less than that obtained at 37 degrees C. When the periplasmic oxidoreductase of E. coli, DsbA, was co-expressed, 69% of the secreted leptin (26% of total proteins) was produced as soluble form at 37 degrees C without the decrease of the amount of leptin produced.

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Does DsbA Have Chaperone-like Activity?

Cited by 43 publications

References 0 publications

Chaperone Activity of DsbC

Chaperone Activity of DsbC

Secretory production of human leptin inEscherichia coli

Secretory production of human leptin in Escherichia coli

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