Chemical and Posttranslational Modification of Escherichia coli Acyl Carrier Protein for Preparation of Dansyl-Acyl Carrier Proteins

2000 ◽  
Vol 20 (2) ◽  
pp. 274-284 ◽  
Author(s):  
Jeffrey A. Haas ◽  
Melissa A. Frederick ◽  
Brian G. Fox
Keyword(s):  
Escherichia Coli ◽  
Posttranslational Modification ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Carrier Proteins ◽  
Acyl Carrier Proteins

scholarly journals Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing

1993 ◽  
Vol 294 (2) ◽  
pp. 521-527 ◽  
Author(s):  
S A Morris ◽  
W P Revill ◽  
J Staunton ◽  
P F Leadlay
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Acyl Carrier Protein ◽  
Saccharopolyspora Erythraea ◽  
Carrier Protein ◽  
Freezing And Thawing ◽  
Acyl Carrier Proteins ◽  
Expression Plasmid ◽  
E Coli ◽  
Protein Dimer

Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer. Electrospray mass spectrometry was used to confirm that the recombinant S. erythraea acyl-carrier protein over-expressed in E. coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct. The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography. The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S. erythraea.

scholarly journals Resin supported acyl carrier protein labeling strategies

RSC Advances ◽  
2014 ◽  
Vol 4 (18) ◽  
pp. 9092-9097 ◽  
Author(s):  
Michael Rothmann ◽  
Nicolas M. Kosa ◽  
Michael D. Burkart
Keyword(s):  
Acyl Carrier Protein ◽  
Protein Labeling ◽  
Carrier Protein ◽  
Carrier Proteins ◽  
Acyl Carrier Proteins ◽  
Functional Modification ◽  
Labeling Strategies

The post-translational modifying enzymes phophopantetheinyl transferase and acyl carrier protein hydrolase show utility as resin supported conjugates in the functional modification of acyl carrier proteins.

scholarly journals Products of fatty acid synthesis by a particulate fraction from germinating pea (Pisum sativum L.)

1981 ◽  
Vol 199 (1) ◽  
pp. 221-226 ◽  
Author(s):  
J Sanchez ◽  
J L Harwood
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Pisum Sativum ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Simultaneous Increase ◽  
Carrier Protein ◽  
Carrier Proteins ◽  
Acyl Carrier Proteins ◽  
Malonyl Coa

The synthesis of lipids and acyl thioesters was studied in microsomal preparations from germinating pea (Pisum sativum cv. Feltham First) seeds. Under conditions of maximal synthesis (in the presence of exogenous acyl-carrier protein) acyl-acyl-carrier proteins accounted for about half the total incorporation from [14C]malonyl-CoA. Decreasing the concentrations of exogenous acyl-carrier protein lowered the overall synthesis of fatty acids by decreasing, almost exclusively, the radioactivity associated with acyl-acyl-carrier proteins. A time-course experiment showed that acyl-acyl-carrier proteins accumulated most of the radioactive label at the beginning of the incubation but, eventually, the amount of radioactivity in that fraction decreased, while a simultaneous increase in the acyl-CoA and lipid fractions was noticed. Addition of exogenous CoA (1 mM) produced a decrease of total incorporation, but an increase in the radioactivity incorporated into acyl-CoA. The microsomal preparations synthesized saturated fatty acids up to C20, including significant proportions of pentadecanoic acid and heptadecanoic acid. Synthesis of these ‘odd-chain’ fatty acids only took place in the microsomal fraction. In contrast, when the 18,000g supernatant (containing the microsomal and soluble fractions) was incubated with [14C]malonyl-CoA, the radioactive fatty acid and acyl classes closely resembled the patterns produced by germinating in the presence of [14C]acetate in vivo. The results are discussed in relation to the role of acyl thioesters in the biosynthesis of plant lipids.

Acyl carrier protein from E. coli. Structural characterization of short-chain acylated acyl carrier proteins by NMR

Biochemistry ◽  
1985 ◽  
Vol 24 (26) ◽  
pp. 7834-7838 ◽  
Author(s):  
Kevin H. Mayo ◽  
J. H. Prestegard
Keyword(s):  
Structural Characterization ◽  
Acyl Carrier Protein ◽  
Short Chain ◽  
Carrier Protein ◽  
Carrier Proteins ◽  
Acyl Carrier Proteins ◽  
E Coli

scholarly journals The Stability of Acyl Carrier Protein in Escherichia coli

1973 ◽  
Vol 248 (12) ◽  
pp. 4461-4466
Author(s):  
Gary L. Powell ◽  
Michael Bauza ◽  
Allan R. Larrabee
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
The Stability

scholarly journals Mutants of Escherichia coli with Temperature-sensitive Malonyl Coenzyme A-Acyl Carrier Protein Transacylase

1974 ◽  
Vol 249 (23) ◽  
pp. 7468-7475
Author(s):  
Mark E. Harder ◽  
Ruth C. Ladenson ◽  
Steven D. Schimmel ◽  
David F. Silbert
Keyword(s):  
Escherichia Coli ◽  
Coenzyme A ◽  
Acyl Carrier Protein ◽  
Temperature Sensitive ◽  
Carrier Protein

scholarly journals Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Protein ◽  
Target Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Carrier Proteins ◽  
E Coli ◽  
Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

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