CiteULike: robert's cloning

Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant tobacco etch virus protease in Escherichia coli BL21(DE3).

RB Kapust — 2008-04-15T14:45:50-00:00

Protein expression and purification, Vol. 24, No. 1. (February 2002), pp. 61-70.

Due to its high degree of sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV protease) is a useful reagent for cleaving genetically engineered fusion proteins. However, the overproduction of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility. Here we demonstrate that the low yield can be attributed to the presence of arginine codons in the TEV protease coding sequence that are rarely used in E. coli and specifically to a tandem pair of AGA codons. The yield of protease can be improved by replacing these rare arginine codons with synonymous ones or by increasing the supply of cognate tRNA that is available to the cell. Furthermore, we show that when ribosomes become stalled at rare arginine codons in the TEV protease mRNA, the nascent polypeptides are targeted for proteolytic degradation in BL21(DE3) cells by a mechanism that does not involve tmRNA-mediated peptide tagging.

Inhibition of tobacco etch virus protease activity by detergents.

AK Mohanty — 2008-04-15T14:41:21-00:00

Protein expression and purification, Vol. 27, No. 1. (January 2003), pp. 109-114.

Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.

Structural basis for the substrate specificity of tobacco etch virus protease.

J Phan — 2008-04-15T14:39:42-00:00

The Journal of biological chemistry, Vol. 277, No. 52. (27 December 2002), pp. 50564-50572.

Because of its stringent sequence specificity, the 3C-type protease from tobacco etch virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2- and 1.8-A resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.

pREP4-sfp Construction

2008-03-27T11:55:10-00:00

Overexpression in Escherichia coli of the AT-rich trpA and trpB genes from the hyperthermophilic archaeon Pyrococcus furiosus.

M Ishida — 2006-10-16T08:35:17-00:00

FEMS Microbiol Lett, Vol. 216, No. 2. (5 November 2002), pp. 179-183.

Expression of AT-rich genes from microorganisms such as archaea is often inefficient in Escherichia coli. The trpA and trpB genes encoding the tryptophan synthase subunits were cloned from the hyperthermophilic archaeon Pyrococcus furiosus. No apparent difference in codon bias was found between the genes. However, using a conventional cloning vector having the lac promoter, the trpB gene was expressed poorly in E. coli, whereas the trpA gene was overexpressed. The expression of the trpB gene was remarkably enhanced (>12-fold) by the introduction of an overlapping leader open reading frame. The expression of the trpA gene was also improved ( approximately 1.5-fold). This approach may be useful for overexpressing various kinds of AT-rich genes.

Effective structure of a leader open reading frame for enhancing the expression of GC-rich genes.

M Ishida — 2006-10-16T08:31:55-00:00

J Biochem (Tokyo), Vol. 132, No. 1. (July 2002), pp. 63-70.

To overexpress broad kinds of GC-rich genes in Escherichia coli, we examined how the structures of leader open reading frames (leader ORFs) affect the expression of GC-rich genes, such as polA, trpA, and trpB, from Thermus thermophilus. When a leader ORF overlapped with the polA-initiation codon by 1 bp in the TGATG motif, gene expression increased by more than 3-fold compared to when a leader ORF was several-bp distant from the initiation codon. A 4-bp overlap with the ATGA motif was more effective than a 1-bp overlap with the TGATG motif. When a 4-bp overlapping leader ORF was placed in front of the successive trpB and trpA genes, the trpA gene was poorly expressed whereas the trpB gene was overexpressed. Mutation analysis revealed that the expression of the trpA gene was strongly enhanced by replacing G and C in the translation termination region of the leader ORF with A and T. In contrast, other mutations, such as alterations between synonymous codons in the trpA-coding region, produced diminished gene expression. Using the most effective leader ORF obtained from these results, new expression vectors were constructed.

GFX PCR DNA and Gel Band Purification Kit

Internetseite Amersham — 2006-09-20T16:19:41-00:00

Scalable recovery of plasmid DNA based on aqueous two-phase separation.

A Frerix — 2006-09-11T15:00:11-00:00

Biotechnol Appl Biochem, Vol. 42, No. Pt 1. (August 2005), pp. 57-66.

Future developments in gene therapy and DNA vaccination depend on cost-effective large-scale production of pharmaceutical-grade pDNA (plasmid DNA). Given the large amount of impurities present in the feedstock, purification processes that have high specificity and capacity at a moderate cost are required. In the present study, we describe a non-chromatographic procedure based on aqueous two-phase extraction allowing a fast and simply scalable capture step. PEG [poly(ethylene glycol)] in combination with potassium citrate or potassium phosphate was tested as phase component for extraction. By increasing either PEG or salt concentration, the partitioning of nucleic acids changed from bottom to top phase. Phase systems with a composition of 15% PEG 800 and 20% potassium phosphate at pH 7.0 showed a strong partitioning of pDNA to the bottom phase, linked to a clear decrease in open circular pDNA, while proteins, genomic DNA and RNA remain at the top or at the interphase. A great advantage of the current process is that the complete procedure of lysis, precipitation, clarification and extraction can be performed in a single vessel. The number of denatured and sheared genomic DNAs in a spiking experiment was found to be depleted by more than 99%.

Practical streptomyces genetics

T Kieser — 2006-06-30T09:19:56-00:00

(2000)

Physical analysis of antibiotic-resistance genes from Streptomyces and their use in vector construction.

CJ Thompson — 2006-06-30T09:12:29-00:00

Gene, Vol. 20, No. 1. (November 1982), pp. 51-62.

Restriction endonuclease cleavage maps of five DNA fragments carrying genes for neomycin phosphotransferase and neomycin acetyltransferase (from Streptomyces fradiae), viomycin phosphotransferase (from S. vinaceus), and ribosomal methylases determining resistance to thiostrepton (from S. azureus) and MLS antibiotics (from S. erythreus) are described, together with a map for the SLP1.2 Streptomyces plasmid used to isolate the fragments. Construction of a versatile Streptomyces cloning vector (pIJ61) is reported. pIJ61 carries neomycin phosphotransferase and thiostrepton resistance genes and has unique BamHI and PstI sites which will allow clone recognition by insertional inactivation of neomycin resistance; cloning sites for several other endonucleases are also present. pIJ28, a shuttle vector for Streptomyces and E. coli, carries neomycin resistance and the SLP1.2 and pBR322 replicons.

pRSET A,B,C

Internetseite Invitrogen — 2006-05-23T12:39:50-00:00

(2006)

pMAL Protein Fusion and Purifiation System

Manual Neb — 2006-05-23T11:14:28-00:00

(November 2007)

XL1-Blue Subcloning-Grade Competent Cells

Manual Stratagene — 2006-05-08T13:23:51-00:00

(2004)

Studies on transformation of Escherichia coli with plasmids.

D Hanahan — 2006-05-08T13:05:36-00:00

J Mol Biol, Vol. 166, No. 4. (5 June 1983), pp. 557-580.

Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.

Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron.

X Zhou — 2006-05-08T09:48:59-00:00

Nucleic Acids Res, Vol. 16, No. 10. (25 May 1988), pp. 4341-4352.

Streptomyces lividans DNA contains a modification which makes it susceptible to double-strand cleavage during electrophoresis in buffers contaminated with ferrous iron (which may be present in some batches of EDTA). The cleavage of the DNA is site-specific and the average fragment size resulting from limit digestion of total S. lividans DNA is about 6kb. DNA from Streptomyces coelicolor A3(2) and several other Streptomyces strains, and from E. coli, is not cleaved under the same conditions. A S. lividans mutant has been isolated which lacks the DNA modification. We suspect that many reports of "poor" preparations of S. lividans plasmids may be due to the above effect.

Genetic transformation of Streptomyces globisporus strain 1912: Restriction barrier and plasmid compatibility

AB Matseliukh — 2006-05-08T08:54:53-00:00

Mikrobiol Z, Vol. 63, No. 1. (b 2001), pp. 15-22.

Low efficiency of genetic transformation of protoplasts of different strains of Streptomyces globisporus 1912 by means of DNA preparations of three vectors pIJ487, pGM160 and pWHM4 is explained by presence of the restriction barrier in the recipients. This obstacle can be overcome by the use of modified DNA of the same vectors, isolated from not numerous transformants. The frequency of transformation by such modified vector DNA was increased by two-three orders in comparison with initial DNA, isolated from Streptomyces lividans TK24, loosing restriction-modification system. The vector pCNB4001, containing the replicon of endogeneous plasmid pSG1912-1, effectively transformed the protoplasts of all S. globisporus 1912 strains. Compatibility of pIJ487 and pSG1912-1 plasmids and incompatibility of the latter and pWHM4 was shown.

Molecular Cloning: A Laboratory Manual (3-Volume Set)

J Sambrook — 2005-04-01T06:31:27-00:00

(15 January 2001)

The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity. In this new edition, authors Joe Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology. Handsomely redesigned and presented in new bindings of proven durability, this three-volume work is essential for everyone using today’s biomolecular techniques. The opening chapters describe essential techniques, some well-established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small. These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing. The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein-protein interactions. The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information. As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved.

Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.

RB Kapust — 2006-02-27T10:17:58-00:00

Protein Eng, Vol. 14, No. 12. (December 2001), pp. 993-1000.

Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.

Cloning and characterization of the genes for p-nitrobenzoate degradation from Pseudomonas pickettii YH105.

AV Yabannavar — 2006-02-13T14:43:34-00:00

Appl Environ Microbiol, Vol. 61, No. 12. (December 1995), pp. 4284-4290.

Pseudomonas pickettii YH105 was isolated for its ability to utilize p-nitrobenzoate as the sole source of carbon, nitrogen, and energy. Degradation of p-nitrobenzoate by this strain proceeds through a reductive route as evidenced by the accumulation of ammonia in the culture medium during growth on p-nitrobenzoate. Enzyme assays and high-performance liquid chromatography (HPLC) analysis of culture supernatants indicate that p-nitrobenzoate is degraded through p-hydroxylaminobenzoate and protocatechuate. In order to clone the genes responsible for the initial steps in the catabolic pathway, a cosmid library was constructed with P. pickettii YH105 genomic DNA. The library was screened for clones capable of transforming p-nitrobenzoate to protocatechuate, using a plate assay specific for diphenolic compounds. HPLC analysis of culture supernatants confirmed that the cosmid clones did indeed produce protocatechuate from p-nitrobenzoate. Five positive cosmid clones that possessed this activity were identified. Restriction digests of the cosmid clones indicated that all of the clones had two EcoRI fragments in common (3.9 and 1.0 kb). One of these cosmid clones, designated pGJZ1601, was chosen for further analysis. Subcloning and activity assay experiments localized the genes responsible for the conversion of p-nitrobenzoate to protocatechuate to a 1.4-kb SalI-SphI DNA fragment. Further subcloning experiments localized the gene coding for p-nitrobenzoate reductase, responsible for the first enzymatic step in the catabolic pathway, to a 0.8-kb SalI-ApaI DNA fragment. The gene for the second step in the catabolic pathway, coding for hydroxylaminolyase, was located adjacent to the gene for the p-nitrobenzoate reductase.

pWHM4* vector map

Hki Inhouse — 2006-02-09T09:53:43-00:00

pGEM T and pGEM T easy

Internetseite Promega — 2006-02-09T09:50:56-00:00

New England Biolabs, DNA Sequences and Maps

Internetseite Neb — 2006-02-09T09:37:00-00:00

Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489.

E Schmutz — 2006-02-08T14:56:00-00:00

Microbiology, Vol. 150, No. Pt 3. (March 2004), pp. 641-647.

The biosynthetic gene clusters of the gyrase inhibitors coumermycin A(1) and clorobiocin contain two different resistance genes (gyrB(R) and parY(R)). Both genes code for B subunits of type II topoisomerases. The authors have now overexpressed and purified the encoded proteins, as well as the corresponding A subunits GyrA and ParX. Expression was carried out in Streptomyces lividans in the form of hexahistidine fusion proteins, allowing purification by nickel affinity chromatography. The complex of GyrA and GyrB(R) was found to catalyse ATP-dependent supercoiling of DNA, i.e. to function as a gyrase, whereas the complex of ParX and ParY(R) catalysed ATP-dependent decatenation and relaxation, i.e. the functions of topoisomerase IV (topo IV). This is believed to represent the first topo IV identified in the class of actinobacteria, and the first demonstration of the formation of a topo IV as a resistance mechanism of an antibiotic producer.

Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus).

J Vara — 2006-02-08T14:53:32-00:00

J Bacteriol, Vol. 171, No. 11. (November 1989), pp. 5872-5881.

Genes that govern the formation of deoxysugars or their attachment to erythronolide B and 3 alpha-mycarosyl erythronolide B, intermediates of the biosynthesis of the 14-membered macrolide antibiotic erythromycin, were cloned from Saccharopolyspora erythraea (formerly Streptomyces erythreus). Segments of DNA that complement the eryB25, eryB26, eryB46, eryC1-60, and eryD24 mutations blocking the formation of erythronolide B or 3 alpha-mycarosyl erythronolide B, when cloned in Escherichia coli-Streptomyces shuttle cosmids or plasmid vectors that can transform S. erythraea, were located in a ca. 18-kilobase-pair region upstream of the erythromycin resistance (ermE) gene. The eryC1 gene lies just to the 5' side of ermE, and one (or possibly two) eryB gene is approximately 12 kilobase pairs farther upstream. Another eryB gene may be in the same region, while an additional eryB mutation appears to be located elsewhere. The eryD gene lies between the eryB and eryC1 genes and may regulate their function on the basis of the phenotype of an EryD- mutant.