High-level secretion of human α1-antitrypsin from Saccharomyces cerevisiae using inulinase signal sequence

Kang, Hyun Ah; Nam, Soo Woo; Chung, Bong Hyun; Yu, Myeong Hee

doi:10.1016/0168-1656(96)01391-0

Cited by 37 publications

(35 citation statements)

References 22 publications

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“…4). 있었고 β-1,3-glucanase 유전자의 발현에 INU1 분비서열이 아주 효과적임을 알 수 있었다 [23][24][25]. 이들 형질전환체에서 의 plasmid 안정성을 확인한 결과 SEY2102에서는 pGInu-exgA plasmid가 76%, pAInu-exgA plasmid가 71% 였으며 2805 에서는 pGInu-exgA plasmid가 55%, pAInu-exgA plasmid 가 66%의 안정성을 보였다 (Table 1) …”

Section: Transformantsunclassified

Optimization for Production of Exo-β-1,3-glucanase (Laminarinase) from Aspergillus oryzae in Saccharomyces cerevisiae

김민정¹,

남수완²,

Tamano³

et al. 2011

KSBB Journal

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1) In this study, a EXGA gene code for exo-β-1,3-glucanase from Aspergillus oryzae was overexpressed and secretory produced in Saccharomyces cerevisiae. To overexpress the β-1,3-glucanase, pGInu-exgA and pAInu-exgA plasmids having GAL10 and ADH1 promoter, respectively, and exoinulinase signal sequence (Inu s.s) were constructed and introduced in S. cerevisiae SEY2102 and 2805. The recombinant β-1,3-glucanase was successfully expressed and secreted into the medium and the β-1,3-glucanase activity in 2102/pGInu-exgA and 2102/pAInu-exgA strain were 5.01 unit/mL and 4.09 unit/mL, respectively. In the 2805/pGInu-exgA and 2805/pAInu-exgA strain, the β-1,3-glucanase activity showed 3.23 unit/mL and 3.22 unit/mL, respectively. Secretory efficiency in each strain reached 95% to 98%. Subsequently, the recombinant β-1,3- Department of Biotechnology and Bioengineering, Pukyong National University, Busan 608-737, Korea glucanase was used for ethanol production. Ethanol productivity in 2102/pAInu-exgA strain was 0.83 g/L when pre-treated Laminaria japonica which has initial reducing sugar of 1.4 g/L was used as substrate. It is assumed that the polysaccharides of Laminaria japonica was effectively saccharified by recombinant β-1,3-glucanase, resulting in increase of ethanol productivity. These results suggested that recombinant β-1,3-glucanase was efficiently overexpressed and secreted in S. cerevisiae SEY2102 as host strain by using ADH1 promoter-Inu s.s system.

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Section: Transformantsunclassified

Optimization for Production of Exo-β-1,3-glucanase (Laminarinase) from Aspergillus oryzae in Saccharomyces cerevisiae

김민정¹,

남수완²,

Tamano³

et al. 2011

KSBB Journal

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“…The cutting area identified by the ycsF endoproteinase was K-R (LysineArginine) amino acid sequence which played a role in peptide signaling in mature Golgi before transported out of the cell. These cutting areas made the K. marxianus inulinase signal peptide had an important role in the secretion of bglp15.2INU protein in S. cerevisiae cells that required for transport across the ER membrane and Golgi body before being transported out of the cell surface (Chung et al, 1995;Kang et al , 1996;Rakestraw et al, 2009;Fitzgerald and Glick, 2014).…”

Section: β-Glucosidasementioning

confidence: 99%

“…The secretion of recombinant β-glucosidase protein without signal peptide was observed in 10 hours induction time because the highest expression of the lux gene on S. cerevisiae by inducing GAL promoter on the pBEVY-GL vector was achieved at the 10 th hour. The gene expression vector and promoter used in this research was same with lux gene research (Chung et al, 1995;Kang et al 1996;Tang et al, 2013;Hong et al, 2015;Gupta et al, 2003). Growth of cells performed on the batch system at some point will stop which can be caused by the reduced substrate required by the cell or due to the accumulation of autotoxic products that inhibited cell growth.…”

Section: β-Glucosidasementioning

confidence: 99%

“…The K. marxianus inulinase signal peptide comprised of 23 amino acids and had three cutting areas recognized by peptidase signal in the endoplasmic reticulum (ER) and one other cutting area 6 th , 7 th , or 8 th amino acids downstream from the ER peptidase signal region and recognized by ycsF endoproteinase (product of the kex2 gene) (Chung et al, 1995;Kang et al, 1996). Three cutting areas recognized by the endoplasmic reticulum peptidase signal were in the order of the S-A-S-V (Serine-Alanine-Serine-Valine) amino acid.…”

Section: β-Glucosidasementioning

confidence: 99%

“…Similarly, most of the human interleukin-2 protein (17 kDa) can be secreted by using K. marxianus inulinase signal peptide. K. marxianus inulinase signal peptide can be used to secrete the protein human α-antitrypsin (52 kDa) up to 70% and inulinase protein by 70% to 90% in S. cerevisiae (Kang et al, 1996;Chung et al, 1995). Because of its high ability to secrete recombinant proteins in S. cerevisiae, the K. marxianus inulinase signal peptide was choosen to secrete the protein β-glucosidase (bglp15.1).…”

Section: Introductionmentioning

confidence: 99%

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Extracellular β-Glucosidase Production from bglp15.2 Gene Carrying Inulinase Signal Peptide in Saccharomyces cerevisiae BY4741

Fitri¹,

Restiawaty

Moeis³

2017

Jurnal Biodjati

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One of the important enzymes in cellulase complex is β-glucosidase. In this research, adding signal peptide of inulinase gene from Kluyveromyces marxianus, cloning, and expressing of bglp15.2 gene in S. cerevisiae BY4741 had been done. Gene of bglp15.2 encoding β-glucosidase has 90% identity to nucleotide sequence of Shewanella frigidimarina NCIMB 400 bacteria. Adding nucleotide sequence of signal peptide was aimed to secrete β-glucosidase and had been done with PCR (Polymerase Chain Reaction) method. The addition of nucleotide sequence of signal peptide in bglp15.2 gene had been done succesfully that indicated from nucleotide sequencing result and the increment of amplicon band size in electroferogram of the last addition PCR step. The bglp15.2 and bglp15.2INU gene (the bglp15.2 gene that has signal peptide nucleotide sequence) were cloned in Escherichia coli DH5α using pGEM-T-Easy vector and pBEVY-GL shuttle vector. The pBEVY-GL shuttle vector was used for transforming S. cerevisiae BY4741 with bglp15.2 and bglp15.2INU. The recombinant S. cerevisiae BY4741 carrying bglp15.2INU gene and growing in 48 hours had extracellularly β-glucosidase enzyme activity of 0,0178 U/ml and the intracellularly activity was 0,0181 U/ml. The β-glucosidase enzyme without signal peptide was not secreted. With K. marxianus inulinase signal peptide, about 50% Bglp15.2INU protein could be secreted. The protein molecular weight of secreted Bglp15.2INU was 44 kDa in SDS-PAGE result.

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Glycosylation of human α1-antitrypsin inSaccharomyces cerevisiae and methylotrophic yeasts

Kang

Sohn

Choi

et al. 1998

Yeast

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Human α1‐antitrypsin (α1‐AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α1‐AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α1‐AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α1‐AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α1‐AT. The human α1‐AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α1‐AT in the methylotrophic yeasts appeared to be relatively shorter than those of α1‐AT in S. cerevisiae. The α1‐AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α1‐AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.

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High-level secretion of human α1-antitrypsin from Saccharomyces cerevisiae using inulinase signal sequence

Cited by 37 publications

References 22 publications

Optimization for Production of Exo-β-1,3-glucanase (Laminarinase) from Aspergillus oryzae in Saccharomyces cerevisiae

Optimization for Production of Exo-β-1,3-glucanase (Laminarinase) from Aspergillus oryzae in Saccharomyces cerevisiae

Extracellular β-Glucosidase Production from bglp15.2 Gene Carrying Inulinase Signal Peptide in Saccharomyces cerevisiae BY4741

Glycosylation of human α1-antitrypsin inSaccharomyces cerevisiae and methylotrophic yeasts

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