[21] Yeast STE 14 methyltransferase, expressed as TrpE-STE 14 fusion protein in Escherichia coli, for in Vitro Carboxylmethylation of prenylated polypeptides

Abstract Objectives: 3,4-Dihydroxybutyric acid (3,4-DHBA) is a multi-functional C4 platform compound with wide applications in the synthesis of materials and pharmaceuticals. Currently, although the biosynthetic pathway for the production of 3,4-DHBA has been developed, low productivity still hampers its use on large scales. Here, a non-natural four-steps biosynthetic pathway was established in recombinant E. coli with a combinatorial strategy.Results: Firstly, several aldehyde dehydrogenases (ALDHs) were screened and characterized for catalyzing the dehydrogenation of 3,4-dihydroxybutanal (3,4-DHB) to 3,4-DHBA through in vitro enzyme assays. Secondly, a recombinant E. coli was successfully constructed to generate 3,4-DHBA from D-xylose by introducing the pathway containing BsGDH, YagF, PpMdlC and ALDH into E. coli with 3.04 g/L 3,4-DHBA obtained. Then, disruption of competing pathways by deleting xylA, ghrA, ghrB and adhP genes contributed to increase the accumulation of 3,4-DHBA by 87%. Final, fusion expression of PpMdlC and YagF resulted in an enhancement of 3,4-DHBA titer (7.71 g/L), as the highest titer reported so far.Conclusions: These results showed that deleting competing pathways and constructing fusion protein could significantly improve the 3,4-DHBA titer in engineered E. coli.

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Overproduction and purification of SulA fusion protein in Escherichia coli and its degradation by Lon protease in vitro

Applied Microbiology and Biotechnology ◽

10.1007/s002530050407 ◽

1995 ◽

Vol 43 (2) ◽

pp. 304-309 ◽

Cited By ~ 10

Author(s):

S. Sonezaki ◽

Y. Ishii ◽

K. Okita ◽

T. Sugino ◽

A. Kondo ◽

...

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Lon Protease

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In vitro reconstitution of the spinach chloroplast cytochrome b6 protein from a fusion protein expressed in Escherichia coli

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/s0167-4838(02)00369-2 ◽

2002 ◽

Vol 1598 (1-2) ◽

pp. 177-184 ◽

Cited By ~ 14

Author(s):

Jarosław Króliczewski ◽

Andrzej Szczepaniak

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Spinach Chloroplast ◽

Cytochrome B6 ◽

In Vitro Reconstitution

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Vaccination with Live Escherichia coli ExpressingBrucella abortus Cu/Zn Superoxide Dismutase Protects Mice against Virulent B. abortus

Infection and Immunity ◽

10.1128/iai.67.2.986-988.1999 ◽

1999 ◽

Vol 67 (2) ◽

pp. 986-988 ◽

Cited By ~ 56

Author(s):

Angel A. Oñate ◽

Ramesh Vemulapalli ◽

Edilia Andrews ◽

Gerhardt G. Schurig ◽

Stephen Boyle ◽

...

Keyword(s):

Escherichia Coli ◽

Superoxide Dismutase ◽

Fusion Protein ◽

Vaccine Strain ◽

Brucella Abortus ◽

Proliferative Response ◽

E Coli ◽

The One

ABSTRACT Vaccination of mice with Escherichia coli expressingBrucella Cu/Zn superoxide dismutase (SOD) [E. coli(pBSSOD)] induced a significant level of protection against virulent Brucella abortus challenge, although this level was not as high as the one reached with B. abortusvaccine strain RB51. In addition, vaccination with E. coli(pBSSOD) induced antibodies to Cu/Zn SOD and a strong proliferative response of splenocytes when stimulated in vitro with a thioredoxin-Cu/Zn SOD fusion protein.

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In vitro reconstitution of antimicrobial pathogen activity by expressed recombinant bovine lactoferrin N-terminal peptide in Escherichia coli

Journal of Dairy Research ◽

10.1017/s0022029907002531 ◽

2007 ◽

Vol 74 (2) ◽

pp. 233-238 ◽

Cited By ~ 7

Author(s):

Hongxia Luo ◽

Shangwu Chen ◽

Fazheng Ren ◽

Huiyuan Guo ◽

Shaohua Lin ◽

...

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Expression System ◽

Bovine Liver ◽

Bovine Lactoferrin ◽

Glutathione S Transferase ◽

Recombinant Peptide ◽

Exon 2 ◽

Enzymatic Proteolysis

Recombinant bovine lactoferrin N-terminal polypeptide (rbLF-N) Escherichia coli expression system was constructed and the rbLF-N antimicrobial activity was displayed by enzymatic proteolysis in this study. A 162 bp 5′-terminal fragment of bovine lactoferrin (bLF) gene from bovine liver gDNA was amplified by PCR. The DNA fragment containing exon-2 of the bLF gene was cloned into the expression vector pGEX-4T1 and the glutathione-S-transferase–rbLF-N (GST-rbLF-N) fusion protein was obtained by over-expression in Esch. coli BL21(DE3). After thrombin/pepsin digestion, the rbLF-N was released from the fusion protein. The recombinant peptide was separated and identified by SDS-PAGE, HPLC and LC-MS/MS analysis. A very strong anti-food-born microbial pathogen activity of the rbLF-N peptides was displayed through bio- and kinetic-assays in vitro. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the rbLF-N peptide for bacterial pathogens Staphylococcus aureus, Streptococcus mutans, Esch. coli and Klebsiella pneumoniae were 11·7, 11·7, 11·7, 23·4 μg and 23·4, 11·7, 11·7, 46·4 μg, respectively. This study created a new route for exploring lactoferrin peptide application in food science.

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Production and characterization of recombinant chicken insulin-like growth factor-II from Escherichia coli

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0140079 ◽

1995 ◽

Vol 14 (1) ◽

pp. 79-90 ◽

Cited By ~ 13

Author(s):

Z Upton ◽

G L Francis ◽

K Kita ◽

J C Wallace ◽

F J Ballard

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Genetically Engineered ◽

Binding Studies ◽

Igf Binding Proteins ◽

Igf Receptor

ABSTRACT Recombinant chicken (c)IGF-II has been produced in Escherichia coli after first modifying a plasmid that coded for a human (h)IGF-II fusion protein. The cIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography and refolded. Recombinant cIGF-II was then released from the fusion protein using a genetically engineered serine protease and purified to homogeneity by reverse-phase HPLC. In vitro analysis of recombinant cIGF-II revealed differences between cIGF-II and its human counterpart. Recombinant cIGF-II was less potent than hIGF-II in stimulating protein synthesis in rat myoblasts. This appeared to be due to a decreased affinity for the type-1 IGF receptor. The human and chicken peptides were similar, however, in studies assessing binding to the type-2 IGF receptor and to IGF-binding proteins. Moreover, recombinant cIGF-II and hIGF-II were equipotent in both biological and receptor binding studies in chick embryo fibroblasts, suggesting that there may be a difference between mammalian and avian type-1 IGF receptors.

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Overexpression of TrxA::BjMT2 (Thioredoxin::Metallothionein Type 2 from Brassica juncea) Fusion Protein in Escherichia coli and In Vitro Cleavage by TEV Protease

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.183-185.947 ◽

2011 ◽

Vol 183-185 ◽

pp. 947-951 ◽

Cited By ~ 1

Author(s):

Yu Zhen Song ◽

Ping Ping Li ◽

Ye Jie Du

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Fusion Protein ◽

Brassica Juncea ◽

Tev Protease ◽

C Terminus ◽

Mass Spectrograph ◽

Thiol Reagent

BjMT2 cDNA clone gene isolated from Brassica juncea was constructed in pETM-20 and expressed in E.coli as a TrxA::BjMT2 fusion protein. After affinity chromatography and cleavage from the TrxA domain, pure BjMT2 protein was obtained which strongly reacted with the thiol reagent monobromobimane. The amino acid sequence determined by mass spectrograph revealed the polypeptide contains 80 amino acids and is full of 8 and 6 cysteines with CC, CXC and C-XX-C motifs clustered near -N and -C terminus, respectively.

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