Effect of molecular chaperones on the soluble expression of alginate lyase inE. coli

Shin, Eun-Jung; Park, So-Lim; Jeon, Sung-Jong; Lee, Jin Woo; Kim, Youngtae; Kim, Yeon-Hee; Nam, Soo-Wan

doi:10.1007/bf02932308

Cited by 8 publications

(9 citation statements)

References 25 publications

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“…The protein was purified by Ni-NTA affinity chromatography, and densitometry of a stained SDS-PAGE gel revealed that the purified SCChi-1 was approximately 99% pure. Most of the gene products expressed in E. coli are organized as aggregate insoluble particles known as inclusion bodies (12,13). However, our results indicated that a high yield of H6SCChi-1 soluble protein could be obtained from E. coli.…”

Section: Expression and Purification Of Scchi-1 In E Colimentioning

confidence: 73%

Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli

Cho

Choi²,

Choi

2011

BMB Reports

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The chitinase-producing strain SC081 was isolated from Korean traditional soy sauce and identified as Bacillus atrophaeus based on a phylogenetic analysis of the 16S rDNA sequence and a phenotypic analysis. A gene encoding chitinase from B. atrophaeus SC081 was cloned in Escherichia coli and was named SCChi-1 (GQ360078). The SCChi-1 nucleotide sequences were composed of 1788 base pairs and 596 amino acids, which were 92.6, 89.6, 89.3, and 78.9% identical to those of Bacillus subtilis (ABG57262), Bacillus pumilus (ABI15082), Bacillus amyloliquefaciens (ABO15008), and Bacillus licheniformis (ACF40833), respectively. A recombinant SCChi-1 containing a hexahistidine tag at the amino-terminus was constructed, overexpressed, and purified in E. coli to characterize SCChi-1. H6SCChi-1 revealed a hydrolytic band on zymograms containing 0.1% glycol chitin and showed the highest lytic activity on colloidal chitin and acidic chitosan. The optimal temperature and pH for chitinolytic activity were 50 o C and pH 8.0, respectively. [BMB reports 2011; 44(3) : 193-198]

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Section: Expression and Purification Of Scchi-1 In E Colimentioning

confidence: 73%

Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli

Cho

Choi²,

Choi

2011

BMB Reports

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Section: Methodsmentioning

confidence: 99%

“…Recombinant plasmids, pALP4, pET3d-ApCpnA, pET21a-ApCPnB, and pG-KJE6 were used in this work [11,21]. The alginate lyase gene (aly, 1.19 kb) from P. elyakovii [19] was subcloned into the pET25-vector, resulting in the plasmid pALP4 (6.7 kb) [21]. The hyperthermophilic chaperonin A and B genes (ApCpnA and ApCpnB) from A. pernix K1 was subcloned into the pRSFDeut-1 vector (Invitrogen, USA), resulting in the plasmid ApCpnA (5.4 kb) and ApCpnB (5.4 kb), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The hyperthermophilic chaperonin A and B genes (ApCpnA and ApCpnB) from A. pernix K1 was subcloned into the pRSFDeut-1 vector (Invitrogen, USA), resulting in the plasmid ApCpnA (5.4 kb) and ApCpnB (5.4 kb), respectively. The plasmid pG-KJE6 encoding dnaKdnaJ-grpE and GroES-GroEL of E. coli was also used as a coexpression partner for the active production of alginate lyase [21]. The plasmids, pALP4, ApCpnA, ApCpnB, and pG-KJE8 were transformed into E. coli Rosetta (DE 3) by CaCl2 method, respectively or simultaneously [14].…”

Section: Methodsmentioning

confidence: 99%

“…When the alginate lyase gene (aly) from P. elyakovii was expressed in E. coli, it yielded inactive aggregates known as inclusion bodies [12,21].…”

Section: Introductionmentioning

confidence: 99%

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Coexpression of Alginate Lyase with Hyperthermophilic Archaea Chaperonin in E. coli

Kim¹,

Kim²,

Nam³

2015

Journal of Life Science

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When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii IAM 14594 was expressed in E. coli, most of the gene product expressed was produced as aggregated insoluble particles known as inclusion bodies. In order to produce with an elevated level of a soluble and active form of alginate lyase in E. coli, the hyperthermophilic chaperonins (ApCpnA and ApCpnB) from archaeon Aeropyrum pernix K1 were employed as the coexpression partners. At 25℃ culture temperature, the level of alginate lyase activity was increased from 10.1 unit/g-soluble protein in aly single expression to 83.1 unit/g-soluble protein by coexpressing with ApCpnA and to 100.3 unit/g-soluble protein by coexpressing with ApCpnB. This results indicate that the coexpression of aly with ApCpnA and ApCpnB revealed a marked enhancement, about 8~10 fold, in the production of alginate lyase as a soluble and active form. Based on the results of various examinations on the expression variables, the optimal conditions for the maximal production of alginate lyase were determined as 1.0 mM IPTG for the inducer concentration, 25℃ for the culture temperature after IPTG induction, and ApCpnB for the coexpression partner. The coexpression set in the present report may be useful in the industrial production of functionally or medically important recombinant proteins in E. coli.

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Cloning and expression of a full-length glutamate decarboxylase gene fromLactobacillus brevis BH2

Kim

Shin

Kim

et al. 2007

Biotechnol. Bioprocess Eng.

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Effect of molecular chaperones on the soluble expression of alginate lyase inE. coli

Cited by 8 publications

References 25 publications

Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli

Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli

Coexpression of Alginate Lyase with Hyperthermophilic Archaea Chaperonin in E. coli

Cloning and expression of a full-length glutamate decarboxylase gene fromLactobacillus brevis BH2

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