Incorporation of fluorescently labeled nonnatural amino acids into proteins in an E. coli in vitro translation system

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

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EXPRESSION AND FUNCTIONAL INTERACTION OF CHICKEN RbAp46 POLYPEPTIDE WITH HISTONES, HISTONE DEACETYLASE-1, AND HISTONE ACETYLTRANSFERASE-1

Indonesian Journal of Chemistry ◽

10.22146/ijc.21296 ◽

2013 ◽

Vol 13 (2) ◽

pp. 136-141

Author(s):

Ahyar Ahmad ◽

Harningsih Karim

Keyword(s):

Amino Acids ◽

Histone Acetyltransferase ◽

Single Step ◽

In Vitro Translation ◽

Translation System ◽

Functional Interaction ◽

In Vitro Experiment ◽

Histone Deacetylase 1 ◽

Agarose Beads

In this study, we cloned and sequenced cDNA encoding the chicken p46 polypeptide, RbAp46. The cDNA encoding a protein consists of 424 amino acids is a member of the WD protein family, with seven WD repeat motifs, and exhibits 90.3% identity to RbAp48, and 94.3% identity to the human RbAp46. The RbAp46 fusion protein were synthesized by in vitro translation system and in Escherichia coli under induction by 50 µM IPTG and single step purified with glutathione-Agarose beads, showed that GST-tagged protein of approximately 72 kDa. The in vitro experiment established that RbAp46 interacts with chicken histones, chHDAC-1, and chHAT-1. The in vitro immunoprecipitation experiment, involving truncated mutants of RbAp46, revealed not only that two regions comprising amino acids 33-179 and 375-404 are necessary for its binding to H2B, but also that two regions comprising amino acids 1-32 and 405-424 are necessary for its binding to H4. Furthermore, the GST pulldown affinity assay, involving truncated mutants of RbAp46, revealed that a region comprising amino acids 359-404 binds to chHAT-1 in vitro. Taken together, these results indicate not only that RbAp46 should participate differentially in a number of DNA-utilizing processes through interactions of its distinct regions with histones and chHAT-1, but also that the proper propeller structure of RbAp46 is not necessary for its interaction with chHAT-1.

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Formation and function of bacterial outer membrane proteins- incorporated liposomes using an in vitro translation system

10.21203/rs.3.rs-964946/v1 ◽

2021 ◽

Author(s):

Koki Kamiya

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Lipid Membranes ◽

Outer Membrane Proteins ◽

Sucrose Density Gradient ◽

In Vitro Translation ◽

Translation System ◽

Patch Clamp Technique ◽

E Coli

Abstract Outer membrane proteins (OMPs), located on the outer membrane of gram-negative bacteria, have a β-strand structure and form nanopores, which allow passage of ions, sugars, and small molecules. Recently, OMPs have been used as sensing elements to detect biological molecules. OMPs are normally expressed and purified from E. coli.. Although the cell-free synthesis of OMPs, such as OmpA and OmpG, is achieved in the presence of liposomes and periplasmic chaperones, the amount of OmpA and OmpG incorporated into the nano-sized liposomes is not clear. In this study, after in vitro translation, the incorporation of OmpG into purified nano-sized liposomes, with various lipid compositions, was investigated. Liposomes containing the synthesized OmpG were purified using a stepwise sucrose density gradient. We report that liposomes prepared with the E. coli lipid extract (PE/PG) had the highest amount of OmpG incorporated compared to liposomes with other lipid compositions, as detected by recording the current across the OmpG containing liposomes using the patch clamp technique. This study reveals some of the requirements for the insertion and refolding of OMPs synthesized by the in vitro translation system into lipid membranes, which plays a role in the biological sensing of various molecules.

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Development of a tRNA-dependent in vitro translation system

RNA ◽

10.1017/s1355838201002539 ◽

2001 ◽

Vol 7 (5) ◽

pp. 765-773 ◽

Cited By ~ 13

Author(s):

RICHARD J. JACKSON ◽

SAWSAN NAPTHINE ◽

IAN BRIERLEY

Keyword(s):

In Vitro Translation ◽

Translation System

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Translational and Structural Requirements of the Early Nodulin Gene enod40, a Short-Open Reading Frame-Containing RNA, for Elicitation of a Cell-Specific Growth Response in the Alfalfa Root Cortex

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.354-366.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 354-366 ◽

Cited By ~ 66

Author(s):

Carolina Sousa ◽

Christina Johansson ◽

Celine Charon ◽

Hamid Manyani ◽

Christof Sautter ◽

...

Keyword(s):

Amino Acids ◽

Biological Activity ◽

Rna Structure ◽

Open Reading Frames ◽

In Vitro Translation ◽

Cortical Cells ◽

Root Cortex ◽

Reading Frame ◽

Early Nodulin

ABSTRACT A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin geneenod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5′ and 3′ regions ofenod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

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Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

Canadian Journal of Microbiology ◽

10.1139/m95-010 ◽

1995 ◽

Vol 41 (1) ◽

pp. 75-87 ◽

Cited By ~ 13

Author(s):

Zerlina M. Naczynski ◽

Andrew M. Kropinski ◽

Chris Mueller

Keyword(s):

Pseudomonas Aeruginosa ◽

Heat Shock ◽

Sigma Factor ◽

Oligonucleotide Probe ◽

In Vitro Transcription ◽

Translation System ◽

Amino Acid Homology ◽

E Coli ◽

Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

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Arabidopsis Cell-free Extract, ACE, a New In Vitro Translation System Derived from Arabidopsis Callus Cultures

Plant and Cell Physiology ◽

10.1093/pcp/pcs013 ◽

2012 ◽

Vol 53 (3) ◽

pp. 602-602

Author(s):

K. Murota ◽

Y. Hagiwara-Komoda ◽

K. Komoda ◽

H. Onouchi ◽

M. Ishikawa ◽

...

Keyword(s):

Callus Cultures ◽

In Vitro Translation ◽

Translation System ◽

Cell Free Extract ◽

Arabidopsis Callus

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An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

RNA ◽

10.1261/rna.825008 ◽

2008 ◽

Vol 14 (3) ◽

pp. 593-602 ◽

Cited By ~ 31

Author(s):

V. V. Zeenko ◽

C. Wang ◽

M. Majumder ◽

A. A. Komar ◽

M. D. Snider ◽

...

Keyword(s):

Mammalian Cells ◽

In Vitro Translation ◽

Translation System ◽

Translational Inhibition ◽

Eif2 Phosphorylation

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four-jointed is required for intermediate growth in the proximal-distal axis in Drosophila

Development ◽

10.1242/dev.121.9.2767 ◽

1995 ◽

Vol 121 (9) ◽

pp. 2767-2777 ◽

Cited By ~ 5

Author(s):

J.L. Villano ◽

F.N. Katz

Keyword(s):

Optic Lobe ◽

Regional Growth ◽

Positional Information ◽

In Vitro Translation ◽

Translation System ◽

Regional Pattern ◽

Microsomal Membranes ◽

Position Sensitive ◽

Coordination Function

Genes capable of translating positional information into regulated growth lie at the heart of morphogenesis, yet few genes with this function have been identified. Mutants in the Drosophila four-jointed (fj) gene show reduced growth and altered differentiation only within restricted sectors of the proximal-distal (PD) axis in the leg and wing, thus fj is a candidate for a gene with this coordination function. Consistent with a position-sensitive role, we show that fj is expressed in a regional pattern in the developing leg, wing, eye and optic lobe. The fj gene encodes a novel type II membrane glycoprotein. When the cDNA is translated in an in vitro translation system in the presence of exogenous microsomal membranes, the intralumenal portion of some of the molecules is cleaved, yielding a secreted C-terminal fragment. We propose that fj encodes a secreted signal that functions as a positive regulator of regional growth and differentiation along the PD axis of the imaginal discs.

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