Ribosomal −1 Frameshifting during Decoding ofBacillus subtilis cdd Occurs at the Sequence CGA AAG

ABSTRACT During translation of the Bacillus subtilis cdd gene, encoding cytidine deaminase (CDA), a ribosomal −1 frameshift occurs near the stop codon, resulting in a CDA subunit extended by 13 amino acids. The frequency of the frameshift is approximately 16%, and it occurs both when the cdd gene is expressed from a multicopy plasmid in Escherichia coli and when it is expressed from the chromosomal copy in B. subtilis. As a result, heterotetrameric forms of the enzyme are formed in vivo along with the dominant homotetrameric species. The different forms have approximately the same specific activity. The cdd gene was cloned in pUC19 such that the lacZ′ gene of the vector followed thecdd gene in the −1 reading frame immediately after thecdd stop codon. By using site-directed mutagenesis of thecdd-lacZ′ fusion, it was shown that frameshifting occurred at the sequence CGA AAG, 9 bp upstream of the in-frame cddstop codon, and that it was stimulated by a Shine-Dalgarno-like sequence located 14 bp upstream of the shift site. The possible function of this frameshift in gene expression is discussed.

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IMPROVEMENT OF T-PA PROPERTIES BY MEANS OF SITE DIRECTED MUTAGENESIS

10.1055/s-0038-1643841 ◽

1987 ◽

Author(s):

N Haigwood ◽

E-P Pâques ◽

G Mullenbach ◽

G Moore ◽

L DesJardin ◽

...

Keyword(s):

Amino Acids ◽

Cleavage Site ◽

Specific Activity ◽

Biological Properties ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Mutant Proteins ◽

C Terminus

The clinical relevance of tissue-plasminogen-activator (t-PA) as a potent thrombolytic agent has recently been established. It has however been recognized that t-PA does not fulfill all conditions required for an ideal thrombolytic pharmaceutical agent; for example, its physiological stability and its short half life in vivo necessitate the use of very large clinical doses. We have therefore attempted to develop novel mutant t-PA proteins with improved properties by creating mutants by site-directed mutagenesis in M13 bacteriophage. Seventeen mutants were designed, cloned, and expressed in CHO cells. Modifications were of three types: alterations to glycosylation sites, truncations of the N- or C-termini, and amino acids changes at the cleavage site utilized to generate the two chain form of t-PA. The mutant proteins were analyzed in vitro for specific activity, fibrin dependence of the plasminogen activation, fibrin affinity, and susceptibility to inhibition by PAI.In brief, the results are: 1) some unglycosylated and partially glycosylated molecules obtained by mutagenesis are characterized by several-fold higher specific activity than wild type t-PA; 2) truncation at the C-terminus by three amino acids yields a molecule with increased fibrin specificity; 3) mutations at the cleavage site lead zo a decreased inhibition by PAI; and 4) recombinants of these genes have been constructed and the proteins were shown to possess multiple improved properties. The use of site directed mutagenesis has proved to be a powerful instrument to modulate the biological properties of t-PA.

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In vivo elimination of parental clones in general and site-directed mutagenesis

Journal of Immunological Methods ◽

10.1016/j.jim.2014.12.008 ◽

2015 ◽

Vol 417 ◽

pp. 67-75 ◽

Cited By ~ 7

Author(s):

Erika G. Holland ◽

Felicity E. Acca ◽

Kristina M. Belanger ◽

Mary E. Bylo ◽

Brian K. Kay ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis

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The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

Biological Chemistry ◽

10.1515/hsz-2017-0178 ◽

2017 ◽

Vol 399 (1) ◽

pp. 73-77 ◽

Cited By ~ 7

Author(s):

Monika B. Dolinska ◽

Yuri V. Sergeev

Keyword(s):

Oculocutaneous Albinism ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Multiple Site ◽

Insect Larvae ◽

Melanin Biosynthesis ◽

Human Tyrosinase

AbstractTyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studiedin vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residuesin vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

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Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

Applied and Environmental Microbiology ◽

10.1128/aem.01628-18 ◽

2018 ◽

Vol 84 (23) ◽

Cited By ~ 2

Author(s):

Efstratios Nikolaivits ◽

Maria Dimarogona ◽

Ioanna Karagiannaki ◽

Angelina Chalima ◽

Ayelet Fishman ◽

...

Keyword(s):

Substrate Specificity ◽

Fruits And Vegetables ◽

Homology Model ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Gene Encoding ◽

Content Type ◽

Polyphenol Oxidases ◽

Increased Activity ◽

Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

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Mending mutations by in vivo site-directed mutagenesis

Molecular Medicine Today ◽

10.1016/s1357-4310(98)01253-2 ◽

1998 ◽

Vol 4 (5) ◽

pp. 188-189

Author(s):

Natasha J Caplen

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis

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Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus

Biochemical Journal ◽

10.1042/bj2880117 ◽

1992 ◽

Vol 288 (1) ◽

pp. 117-121 ◽

Cited By ~ 48

Author(s):

E P Ko ◽

H Akatsuka ◽

H Moriyama ◽

A Shinmyo ◽

Y Hata ◽

...

Keyword(s):

Amino Acids ◽

Reaction Mechanism ◽

Amino Acid ◽

Bacillus Pumilus ◽

Specific Activity ◽

Sequence Similarity ◽

Site Directed Mutagenesis ◽

Dimensional Structure ◽

Amino Acid Sequence Similarity ◽

Directed Mutagenesis

To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.

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Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

10.21248/gups.61063 ◽

2021 ◽

Author(s):

◽

Madeleine Huber

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Stop Codon ◽

Haloferax Volcanii ◽

Site Directed Mutagenesis ◽

Start Codon ◽

Directed Mutagenesis ◽

Ribosome Display ◽

E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

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An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.195-196.407 ◽

2012 ◽

Vol 195-196 ◽

pp. 407-411

Author(s):

Mu Qing Qiu

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Recombinant Plasmid ◽

Genomic Dna ◽

Gene Replacement ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

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