scholarly journals Gene synthesis, expression in Escherichia coli, purification and characterization of the recombinant bovine acyl-CoA-binding protein

1991 ◽  
Vol 276 (3) ◽  
pp. 817-823 ◽  
Author(s):  
S Mandrup ◽  
P Højrup ◽  
K Kristiansen ◽  
J Knudsen
Keyword(s):  
Escherichia Coli ◽  
Binding Protein ◽  
Fatty Acid Synthetase ◽  
Cellular Protein ◽  
Exchange Chromatography ◽  
Synthetic Gene ◽  
Binding Characteristics ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Acetyl Group

A synthetic gene encoding the 86 amino acid residues of mature acyl-CoA-binding protein (ACBP), and the initiating methionine was constructed. The synthetic gene was assembled from eight partially overlapping oligonucleotides. Codon usage and nucleotides surrounding the ATG translation-initiation codon were chosen to allow efficient expression in Escherichia coli as well as in yeast. The synthetic gene was inserted into the expression vector pKK223-3 and expressed in E. coli. In maximally induced cultures, recombinant ACBP constitutes 12-15% of total cellular protein. A fraction highly enriched for recombinant ACBP was obtained by extracting induced E. coli cells with 1 M-acetic acid. Recombinant ACBP was purified to homogeneity by successive use of gel-filtration chromatography, ion-exchange chromatography and reverse-phase h.p.l.c. Recombinant ACBP differed from native ACBP by lacking the N-terminal acetyl group. The acyl-CoA-binding characteristics of recombinant ACBP did not differ from those of native ACBP, and the two proteins showed the same ability to induce medium-chain acyl-CoA synthesis by goat mammary-gland fatty acid synthetase. It was concluded that the N-terminal acetyl group is not important for acyl-CoA binding.

Deletion of the Penicillin-Binding Protein 6 Gene of Escherichia coli

1982 ◽  
Vol 152 (2) ◽  
pp. 904-906
Author(s):  
Jennifer K. Broome-Smith ◽  
Brian G. Spratt
Keyword(s):  
Escherichia Coli ◽  
Marked Effect ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
E Coli

A strain of Escherichia coli with a deletion of the penicillin-binding protein 6 gene ( dacC ) has been constructed. The properties of this strain establish that the complete lack of penicillin-binding protein 6 has no marked effect on the growth of E. coli .

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

scholarly journals Analysis of crystalline and solution states of ligand-free spermidine N-acetyltransferase (SpeG) from Escherichia coli

2019 ◽  
Vol 75 (6) ◽  
pp. 545-553 ◽  
Author(s):  
Ekaterina V. Filippova ◽  
Steven Weigand ◽  
Olga Kiryukhina ◽  
Alan J. Wolfe ◽  
Wayne F. Anderson
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Crystal Structures ◽  
Coenzyme A ◽  
Amino Group ◽  
Acetyl Coenzyme A ◽  
E Coli ◽  
Ligand Free ◽  
Acetyl Group ◽  
Terminal Amino

Spermidine N-acetyltransferase (SpeG) transfers an acetyl group from acetyl-coenzyme A to an N-terminal amino group of intracellular spermidine. This acetylation inactivates spermidine, reducing the polyamine toxicity that tends to occur under certain chemical and physical stresses. The structure of the SpeG protein from Vibrio cholerae has been characterized: while the monomer possesses a structural fold similar to those of other Gcn5-related N-acetyltransferase superfamily members, its dodecameric structure remains exceptional. In this paper, structural analyses of SpeG isolated from Escherichia coli are described. Like V. cholerae SpeG, E. coli SpeG forms dodecamers, as revealed by two crystal structures of the ligand-free E. coli SpeG dodecamer determined at 1.75 and 2.9 Å resolution. Although both V. cholerae SpeG and E. coli SpeG can adopt an asymmetric open dodecameric state, solution analysis showed that the oligomeric composition of ligand-free E. coli SpeG differs from that of ligand-free V. cholerae SpeG. Based on these data, it is proposed that the equilibrium balance of SpeG oligomers in the absence of ligands differs from one species to another and thus might be important for SpeG function.

scholarly journals Overproduction of Penicillin-Binding Protein 2 and Its Inactive Variants Causes Morphological Changes and Lysis in Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 4975-4983 ◽  
Author(s):  
Blaine A. Legaree ◽  
Calvin B. Adams ◽  
Anthony J. Clarke
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Morphological Changes ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Prolonged Incubation ◽  
Lytic Transglycosylases ◽  
E Coli ◽  
Derivatives Of

ABSTRACT Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.

scholarly journals The c-Type Cytochrome OmcA Localizes to the Outer Membrane upon Heterologous Expression in Escherichia coli

2008 ◽  
Vol 190 (14) ◽  
pp. 5127-5131 ◽  
Author(s):  
James W. Donald ◽  
Matthew G. Hicks ◽  
David J. Richardson ◽  
Tracy Palmer
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Shewanella Oneidensis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Type Ii Secretion System ◽  
Surface Localization ◽  
K 12

ABSTRACT We have functionally produced the outer membrane cytochrome OmcA from Shewanella oneidensis in Escherichia coli. Substrate accessibility experiments indicate that OmcA is surface exposed in an E. coli B strain but not in a K-12 strain. We show that a functional type II secretion system is required for surface localization.

Peptide nucleic acid restores colistin susceptibility through modulation of MCR-1 expression in Escherichia coli

2020 ◽  
Author(s):  
Xiaoming Wang ◽  
Yao Wang ◽  
Zhuoren Ling ◽  
Chaoyang Zhang ◽  
Mingming Fu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Peptide Nucleic Acid ◽  
Promoter Activity ◽  
Sequence Variation ◽  
Promoter Sequence ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Expression Host ◽  
Antisense Approach ◽  
Increased Susceptibility

Abstract Background Plasmid-mediated mechanisms of drug resistance accelerate the spread of polymyxin resistance, leaving clinicians with few or no antibacterial options for the treatment of infections caused by MDR bacteria, especially carbapenemase-producing strains. Objectives To evaluate the associations among promoter sequence variation, mcr-1 expression, host factors and levels of colistin resistance and to propose antisense agents such as peptide nucleic acids (PNAs) targeting mcr-1 as a tool to restore colistin susceptibility through modulation of MCR-1 expression in Escherichia coli. Methods A β-galactosidase assay was performed to study mcr-1 promoter activity. Quantitative real-time PCR and western blot assays were used to identify the expression level of MCR-1 in WT strains and transformants. Three PNAs targeting different regions of mcr-1 were designed and synthesized to determine whether they can effectively inhibit MCR-1 expression. MIC was measured to test colistin susceptibility in the presence or absence of PNA-1 in mcr-1-carrying E. coli. Results Variation in the mcr-1 promoter sequence and host species affect promoter activity, MCR-1 expression levels and colistin MICs. One PNA targeting the ribosome-binding site fully inhibited the expression of mcr-1 at a concentration of 4 μM, resulting in significantly increased susceptibility to colistin. The MIC90 of colistin decreased from 8 to 2 mg/L (P < 0.05) in the presence of 4 μM PNA. Conclusions These findings suggest that the antisense approach is a possible strategy to combat mcr-1-mediated resistance as well as other causes of emerging global resistance.

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