Synthesis of proteins by automated flow chemistry

Ribosomes produce most proteins of living cells in seconds. Here we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids over 328 consecutive reactions. The machine is rapid - the peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.

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Synthesis of Proteins by Automated Flow Chemistry

10.26434/chemrxiv.11833503 ◽

2020 ◽

Author(s):

Nina Hartrampf ◽

Azin Saebi ◽

Mackenzie Poskus ◽

Zachary P. Gates ◽

Alexander J. Callahan ◽

...

Keyword(s):

Amino Acids ◽

Chemical Synthesis ◽

Flow Chemistry ◽

Living Cells ◽

Chain Elongation ◽

High Fidelity ◽

Structural Units ◽

Synthetic Materials ◽

Consecutive Reactions ◽

Fast Flow

Ribosomes produce most proteins of living cells in seconds. Here we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids over 328 consecutive reactions. The machine is rapid - the peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.

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Studies on Chromatographic Fractioning by Cations Exchangers of a Bovine Hemoglobin Hydrolysate

Revista de Chimie ◽

10.37358/rc.18.10.6626 ◽

2018 ◽

Vol 69 (10) ◽

pp. 2794-2798

Author(s):

Alina Diana Panainte ◽

Ionela Daniela Morariu ◽

Nela Bibire ◽

Madalina Vieriu ◽

Gladiola Tantaru ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Retention Time ◽

Chromatographic Separation ◽

Bovine Hemoglobin ◽

Reverse Phase ◽

Hplc System ◽

Fast Flow ◽

Hydrolysis Of ◽

Phase Column

A peptidic hydrolysate has been obtained through hydrolysis of bovine hemoglobin using pepsin. The fractioning of the hydrolysate was performed on a column packed with CM-Sepharose Fast Flow. The hydrolysate and each fraction was filtered and then injected into a HPLC system equipped with a Vydak C4 reverse phase column (0.46 x 25 cm), suitable for the chromatographic separation of large peptides with 20 to 30 amino acids. The detection was done using mass spectrometry, and the retention time, size and distribution of the peptides were determined.

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Flow chemistry approaches directed at improving chemical synthesis

Green Processing and Synthesis ◽

10.1515/gps-2013-0029 ◽

2013 ◽

Vol 2 (3) ◽

Cited By ~ 16

Author(s):

Ian R. Baxendale ◽

Laurens Brocken ◽

Carl J. Mallia

Keyword(s):

Chemical Synthesis ◽

Flow Chemistry

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Chemical synthesis and enzymatic, stereoselective hydrolysis of a functionalized dihydropyrimidine for the synthesis of β-amino acids

AMB Express ◽

10.1186/s13568-015-0174-8 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Christin Slomka ◽

Sabilla Zhong ◽

Anna Fellinger ◽

Ulrike Engel ◽

Christoph Syldatk ◽

...

Keyword(s):

Amino Acids ◽

Chemical Synthesis ◽

Stereoselective Hydrolysis ◽

Hydrolysis Of

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A Straightforward Methodology to Overcome Solubility Challenges for N-Terminal Cysteinyl Peptide Segments Used in Native Chemical Ligation

10.26434/chemrxiv.13174922 ◽

2020 ◽

Author(s):

Skander Abboud ◽

El hadji Cisse ◽

Michel Doudeau ◽

Hélène Bénédetti ◽

Vincent AUCAGNE

Keyword(s):

Amino Acids ◽

Chemical Synthesis ◽

Building Blocks ◽

Native Chemical Ligation ◽

Synthetic Approach ◽

Chemical Ligation ◽

Limited Solubility ◽

Peptide Segments

One of the main limitations encountered during the chemical synthesis of proteins through native chemical ligation (NCL) is the limited solubility of some of the peptide segments. The most commonly used solution to overcome this problem is to derivatize the segment with a temporary solubilizing tag. Conveniently, the tag can be introduced on the thioester segment in such a way that it is removed concomitantly with the NCL reaction. We herein describe a generalization of this approach to N-terminal cysteinyl segment counterparts, using a straightforward synthetic approach that can be easily automated from commercially available building blocks, and applied it to a well-known problematic target, SUMO-2 (93 amino acids).

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Effect of Lime and Soil Conditioners on Crop Yields and Soil Aggregation

The Journal of Agriculture of the University of Puerto Rico ◽

10.46429/jaupr.v41i3.12617 ◽

1969 ◽

Vol 41 (3) ◽

pp. 179-188

Author(s):

M. A. Lugo-López ◽

J. A. Bonnet ◽

R. Pérez-Escolar

Keyword(s):

Large Scale ◽

Crop Yields ◽

Acid Soils ◽

Aggregate Stability ◽

Soil Conditions ◽

Structural Units ◽

Ratoon Crop ◽

Synthetic Materials ◽

Soil Conditioners ◽

Plant Crop

Data are presented here on the effect of synthetic soil conditioners on aggregation and aggregate stability of acid Lares clay and on their effect, with or without lime, on the yields of sweetpotatoes, cotton, and corn. Three conditioners were used: Formulations 6 and 9 of Krilium, and Aerotil, dry form, each at the rates of 900, 1,800, and 3,600 pounds to the acre. There were 20 treatments: Check, lime, conditioners at three levels, and conditioners at the same three levels plus lime. The data presented indicate that these conditioners will stabilize soil structural units, but will not form them. Five crops were grown as a sequence: Sweetpotatoes, cotton, cotton (a ratoon crop), sweetpotatoes, and corn. All crops, except the cotton ratoon, showed some response to the application of soil conditioners. Sweetpotato, a root crop, was more responsive; but the cotton plant crop responded also to stabilized good structural soil conditions. The largest crop responses measured were in the limed treatments. Increases attributable to lime were obtained either in the presence or absence of synthetic soil conditioners. Liming and rational fertilization seems to be the key to increased productivity in some acid soils of Puerto Rico. The synthetic materials do not have practical possibilities in large-scale farming.

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Catalytic Methods in Flow Chemistry

Catalysts ◽

10.3390/catal9080663 ◽

2019 ◽

Vol 9 (8) ◽

pp. 663

Author(s):

Christophe Len ◽

Renzo Luisi

Keyword(s):

Chemical Synthesis ◽

Continuous Flow ◽

Flow Chemistry ◽

Pharmaceutical Companies ◽

Enabling Technology ◽

Continuous Flow Chemistry ◽

The Way

Continuous flow chemistry is radically changing the way of performing chemical synthesis, and several chemical and pharmaceutical companies are now investing in this enabling technology [...]

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ChemInform Abstract: NUCLEIC ACID COMPONENTS AND THEIR ANALOGUES PART 163, AMINO ACIDS AND PEPTIDES PART 119, S-(PYRIMIDIN-2-YL)-L-CYSTEINE, CHEMICAL SYNTHESIS AND BIOSYNTHESIS IN ESCHERICHIA COLI

Chemischer Informationsdienst ◽

10.1002/chin.197418437 ◽

1974 ◽

Vol 5 (18) ◽

pp. no-no

Author(s):

A. HOLY ◽

I. VOTRUBA ◽

K. JOST

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Nucleic Acid ◽

Chemical Synthesis

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A Novel mTOR-Regulated Phosphorylation Site in Elongation Factor 2 Kinase Modulates the Activity of the Kinase and Its Binding to Calmodulin

Molecular and Cellular Biology ◽

10.1128/mcb.24.7.2986-2997.2004 ◽

2004 ◽

Vol 24 (7) ◽

pp. 2986-2997 ◽

Cited By ~ 178

Author(s):

Gareth J. Browne ◽

Christopher G. Proud

Keyword(s):

Amino Acids ◽

Protein Kinase ◽

Phosphorylation Site ◽

Elongation Factor ◽

Mtor Pathway ◽

Mtor Signaling ◽

Chain Elongation ◽

Binding Motif ◽

Elongation Factor 2 ◽

Eef2 Kinase

ABSTRACT Eukaryotic elongation factor 2 (eEF2) kinase is an unusual calcium- and calmodulin-dependent protein kinase that is regulated by insulin through the rapamycin-sensitive mTOR pathway. Here we show that insulin decreases the ability of eEF2 kinase to bind calmodulin in a rapamycin-sensitive manner. We identify a novel phosphorylation site in eEF2 kinase (Ser78) that is located immediately next to its calmodulin-binding motif. Phosphorylation of this site is increased by insulin in a rapamycin-sensitive fashion. Regulation of the phosphorylation of Ser78 also requires amino acids and the protein kinase phosphoinositide-dependent kinase 1. Mutation of this site to alanine strongly attenuates the effects of insulin and rapamycin both on the binding of calmodulin to eEF2 kinase and on eEF2 kinase activity. Phosphorylation of Ser78 is thus likely to link insulin and mTOR signaling to the control of eEF2 phosphorylation and chain elongation. This site is not a target for known kinases in the mTOR pathway, e.g., the S6 kinases, implying that it is phosphorylated by a novel mTOR-linked protein kinase that serves to couple hormones and amino acids to the control of translation elongation. eEF2 kinase is thus a target for mTOR signaling independently of previously known downstream components of the pathway.

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