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1.
Biomol NMR Assign ; 14(1): 141-146, 2020 04.
Article in English | MEDLINE | ID: mdl-32052266

ABSTRACT

CanA from Pyrodictium abyssi forms a heat-resistant organic hollow-fiber network together with CanB and CanC. An N-terminally truncated construct of CanA (K1-CanA) gave NMR spectra of good quality that could be assigned by three-dimensional NMR methods on 15N and 13C-15N enriched protein. We assigned the chemical shifts of 96% of all backbone 1HN atoms, 98% of all backbone 15N atoms, 100% of all 13Cα atoms, 100% of all 1Hα atoms, 90% of all 13C' atoms, and 100% of the 13Cß atoms. Two short helices and 10 ß-strands are estimated from an analysis of the chemical shifts leading to a secondary structure content of K1-CanA of 6% helices, 44% ß-pleated sheets, and 50% coils.


Subject(s)
Archaea/metabolism , Archaeal Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular , Amino Acid Sequence , Archaeal Proteins/isolation & purification , Peptides/chemistry , Protein Structure, Secondary , Proteolysis
2.
Protein Expr Purif ; 82(1): 26-31, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22100525

ABSTRACT

Cell-free protein synthesis is a promising technology featuring many advantages compared to in vivo expression techniques. However, most proteins are still synthesized in vivo due to relatively low protein yields commonly achieved in vitro, especially in the batch mode of reaction. In Escherichia coli S30 extract-based cell-free systems protein yields are supposed to be partially limited by a secondary structure formation of the mRNA. In this study we checked promising members of various classes of RNA chaperones and several different RNA helicases on their ability to enhance in vitro translation. The data clearly show that the addition of none of these factors provides a general solution to the problem. However, protein yields can be increased in presence of a microRNA hybridizing with the 5' untranslated region of mRNAs, possibly by inducing structural changes improving accessibility of the Shine Dalgarno sequence for the ribosomes.


Subject(s)
Cell-Free System/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Protein Biosynthesis , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , Escherichia coli/chemistry , Escherichia coli/enzymology , MicroRNAs/metabolism , Nucleic Acid Conformation , RNA Helicases/metabolism , RNA, Bacterial/chemistry , RNA, Messenger/chemistry
3.
J Biotechnol ; 150(1): 44-50, 2010 Oct 01.
Article in English | MEDLINE | ID: mdl-20638424

ABSTRACT

Cell-free production of proteins is a rapid developing technology for many applications in proteomic sciences. Although great progress was made in the last years, in vitro protein synthesis is still less efficient than in vivo protein synthesis. For Escherichia coli based systems, all factors needed for cell-free protein synthesis are established, as shown in the reconstituted PURE system. Here we report the influence of additional translation factors, aminoacyl-tRNA synthetases and translational active ribosomes on cell-free protein synthesis of E. coli S30 extracts, indicating that none of these factors is a limiting factor for the protein synthesis. Furthermore, we show that varying the ratio of ribosomes including associated factors to other factors present in the extracts could not increase the yield of protein synthesized. In summary our results provide strong evidence for an optimal reconstitution of the translation machinery in E. coli S30 extracts.


Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Cell-Free System/metabolism , Protein Biosynthesis , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Amino Acyl-tRNA Synthetases/metabolism , Cell Extracts , Cell-Free System/chemistry , Escherichia coli/chemistry , Escherichia coli/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism
5.
FEBS J ; 272(18): 4691-702, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16156790

ABSTRACT

Cold shock proteins (CSPs) form a family of highly conserved bacterial proteins capable of single-stranded nucleic acid binding. They are suggested to act as RNA chaperones during cold shock inhibiting the formation of RNA secondary structures, which are unfavourable for transcription and translation. To test this commonly accepted theory, isolated CSPs from a mesophilic, thermophilic and a hyperthermophilic bacterium (Bacillus subtilis, Bacillus caldolyticus and Thermotoga maritima) were studied in an Escherichia coli based cell free expression system on their capability of enhancing protein expression by reduction of mRNA secondary structures. The E. coli based expression of chloramphenicol acetyltransferase and of H-Ras served as model systems. We observed a concentration-dependent suppression of transcription and translation by the different CSPs which makes the considered addition of CSPs for enhancing the protein expression in in vitro translation systems obsolete. Protein expression was completely inhibited at CSP concentrations present under cold shock conditions. The CSP concentrations necessary for 50% inhibition were lowest (140 microm) for the protein of the hyperthermophilic and increased when the thermophilic (215 microm) or even the mesophilic protein (451 microm) was used. Isolated in vitro transcription under the influence of CSPs showed that the transcriptory effect is independent from the rest of the cell. It could be shown in a control experiment that the inhibition of protein expression can be removed by addition of hepta-2'-desoxy-thymidylate (dT7); a heptanucleotide that competitively binds to CSP. The data are in line with a hypothesis that CSPs act on bulk protein expression not as RNA chaperones but inhibit their transcription and translation by rather unspecific nucleic acid binding.


Subject(s)
Bacterial Proteins/pharmacology , Cold Temperature , Heat-Shock Proteins/pharmacology , Molecular Chaperones/pharmacology , Protein Biosynthesis/drug effects , Transcription, Genetic/drug effects , Bacillus/chemistry , Chloramphenicol O-Acetyltransferase , Gene Expression Regulation/drug effects , Genes, ras , Nucleic Acid Conformation/drug effects , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA-Binding Proteins/pharmacology , Thermotoga maritima/chemistry
6.
Biol Chem ; 385(3-4): 303-13, 2004.
Article in English | MEDLINE | ID: mdl-15134345

ABSTRACT

The central region of the matrix protein p17 of HIV-1 is known to be essential during virus assembly. We substituted alanines for amino acid triplets in this region of p17 (amino acid residues 47 to 55: NPG LLE TSE). Introduction of the respective mutations into the gag-coding sequence of HI-proviruses and subsequent transfection into Cos-7 cells led to particle production and release. Exchange of LLE resulted in the production of non-infectious particles. These residues may be important for correct folding and assembly of the processed matrix protein and the production of infectious HIV. In vitro studies of wild-type and mutated matrix proteins using spectroscopic methods (NMR, fluorescence, CD) yielded detailed data about structure and stability. Two-dimensional NMR spectroscopy showed that wild-type and mutant proteins (p17-NPG and p17-TSE) are well folded. Besides structural changes at the mutated site, chemical shift changes indicate small but significant long range structural rearrangements. The stability against chemically and thermally induced unfolding of the mutants p17-NPG and p17-TSE was slightly decreased, while that of p17-LLE was drastically diminished. The alterations have only a local effect on protein folding for the mutants p17-NPG and p17-TSE, and the globular tertiary structure remains nearly unchanged. For p17-LLE, however, the substitutions seem to trigger significant changes in structural elements.


Subject(s)
HIV Infections/metabolism , HIV-1/genetics , HIV-1/pathogenicity , Viral Matrix Proteins/biosynthesis , Viral Matrix Proteins/genetics , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , HIV Infections/genetics , HIV Infections/virology , HIV-1/chemistry , Humans , Molecular Sequence Data , Protein Folding , Protein Structure, Tertiary/physiology , Viral Matrix Proteins/chemistry , Virion/metabolism
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