Characterization of TRAP-Mediated Regulation of the B. subtilis trp Operon Using In Vitro Transcription and Transcriptional Reporter Fusions In Vivo

ABSTRACT Bacteriophage GA-1, which infects Bacillus sp. strain G1R, is evolutionarily related to phage φ29, which infectsBacillus subtilis. We report the characterization of several GA-1 promoters located at either end of its linear genome. Some of them are unique for GA-1 and drive the expression of open reading frames that have no counterparts in the genome of φ29 or related phages. These unique promoters are active at early infection times and are repressed at late times. In vitro transcription reactions revealed that the purified GA-1-encoded protein p6 represses the activity of these promoters, although the amount of p6 required to repress transcription was different for each promoter. The level of protein p6 produced in vivo increases rapidly during the first stage of the infection cycle. The protein p6 concentration may serve to modulate the expression of these early promoters as infection proceeds.

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Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

10.1101/815092 ◽

2019 ◽

Author(s):

Monica Z. Wu ◽

Haruichi Asahara ◽

George Tzertzinis ◽

Bijoyita Roy

Keyword(s):

High Temperature ◽

In Vitro Transcription ◽

Synthesis Process ◽

Run Off ◽

Post Synthesis ◽

Cellular Immune ◽

By Products

ABSTRACTThe use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitro-transcribed mRNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo, that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products—one arising from 3’ extension of the run-off product and one formed by the production of antisense RNAs—and demonstrate that at a high temperature, T7 RNAP has reduced 3’-self extension of the run-off product. We show that template-encoded poly-A tailing does not affect 3’-self extension but reduces the formation of the antisense RNA by-products and that combining high-temperature IVT with template-encoded poly-A tailing prevents formation of both kinds of by-products.

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Expression of Murine Coronavirus Recombinant Papain-Like Proteinase: Efficient Cleavage Is Dependent on the Lengths of both the Substrate and the Proteinase Polypeptides

Journal of Virology ◽

10.1128/jvi.73.4.2658-2666.1999 ◽

1999 ◽

Vol 73 (4) ◽

pp. 2658-2666 ◽

Cited By ~ 20

Author(s):

Henry Teng ◽

Josefina D. Piñón ◽

Susan R. Weiss

Keyword(s):

Hepatitis Virus ◽

In Vitro Transcription ◽

Proteolytic Processing ◽

Replicase Gene ◽

Cleavage Activity ◽

Viral Sequences ◽

Rna Polymerase Promoter

ABSTRACT Proteolytic processing of the replicase gene product of mouse hepatitis virus (MHV) is essential for viral replication. In MHV strain A59 (MHV-A59), the replicase gene encodes two predicted papain-like proteinase (PLP) domains, PLP-1 and PLP-2. Previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cisand trans cleavage activities for PLP-1. We have cloned and overexpressed the PLP-1 domain in Escherichia coli by using a T7 RNA polymerase promoter system or as a maltose-binding protein (MBP) fusion protein. With both overexpression systems, the recombinant PLP-1 exhibited trans cleavage activity when incubated with in vitro-synthesized viral polypeptide substrates. Subsequent characterization of the recombinant PLP-1 revealed that in vitrotrans cleavage is more efficient at 22°C than at higher temperatures. Using substrates of increasing lengths, we observed efficient cleavage by PLP-1 requires a substrate greater than 69 kDa. In addition, when PLP-1 was expressed as a polypeptide that included additional viral sequences at the carboxyl terminus of the predicted PLP-1 domain, a fivefold increase in proteolytic activity was observed. The data presented here support previous data suggesting that in vitro and in vivo cleavage of the ORF 1a polyprotein by PLP-1 can occur in both in cis and in trans. In contrast to the cleavage activity demonstrated for PLP-1, no in vitro cleavage incis or in trans could be detected with PLP-2 expressed either as a polypeptide, including flanking viral sequences, or as an MBP fusion enzyme.

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Characterization of an Endogenous Transcription Inhibitor from Physarum polycephalum

Zeitschrift für Naturforschung C ◽

10.1515/znc-1979-1-216 ◽

1979 ◽

Vol 34 (1-2) ◽

pp. 76-86 ◽

Cited By ~ 4

Author(s):

Armin Hildebrandt ◽

Rudolf Mengel

Keyword(s):

Rna Polymerase ◽

Physarum Polycephalum ◽

Gel Filtration ◽

In Vitro Transcription ◽

Exchange Chromatography ◽

Heat Stable ◽

Transcription Inhibitor

Abstract A substance has been purified from isolated nuclei of Physarum polycephalum by equilibrium and velocity gradient centrifugations, ion exchange chromatography and gel filtration which has a high molecular weight, can be labeled in vivo with 32P, is heat stable and resistant to amylases, proteases, nucleases and phosphodiesterase but is sensitive to phosphatases or hydrolysis. This material consists of phospate and glycerol. It selectively inhibits in vitro transcription of RNA polymerases, predominantly the homologous enzyme A by binding to the enzyme. In the presence of this inhibitor of transcription a stable RNA polymerase-template complex cannot be formed. Binding to and inactivation of RNA polymerase is reversible at high ionic strength.

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In vivo and in vitro characterization of immediate release dosage forms containing n-acetylcysteine using the dynamic open flow-through test apparatus

10.26226/morressier.57d6b2b7d462b8028d88dd29 ◽

2016 ◽

Author(s):

Maximilian Sager

Keyword(s):

Immediate Release ◽

Dosage Forms ◽

Test Apparatus ◽

Open Flow ◽

In Vitro Characterization ◽

Flow Through

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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