High level expression of genes cloned in phage λgt11 (original) (raw)
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Phasmid vectors for identification of genes by complementation of Escherichia coli mutants
Journal of bacteriology, 1985
A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, w...
λ ZAP: a bacteriophage λ expression vector within vivoexcision properties
Nucleic Acids Research, 1988
A lambda Insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coBa phagemld, pBtuescript SK(-), contained within the vector, can be excised by 11 or M13 helper phage. The excision process elmlnates the need to subcbne DNA inserts from the lambda phage into a plasmld by restriction digestion and igation. This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the 11 bacteriophage origin of repfication (1). Six of 21 restriction sites In the excised pBluescript SK polyBnker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated and analyzed. The lambda ZAP vector was tested by the preparation of a chicken liver cDNA Hbrary and the isolation of actin clones by screening with oBgonucleotide probes. Putative actln clones were excised from the lambda vector and Identified by DNA sequencing. The ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgQ anti-glucocerbrosidase antibodies.
PLOS ONE, 2020
Cloning and expression of a desired gene is indispensable in molecular biology studies. Expression vectors, in this regard, should offer much needed flexibility and choice of cloning strategies for both in vivo and in vitro protein expression experiments. Furthermore, availability of option to choose from various reporter tags allows one to be flexible during designing of an experiment in a more relevant manner. Thus, the need of a versatile expression system cannot be ignored. Although several different expression vectors are available for gene expression in mycobacteria, they lack the required versatility of expression and the inclusion of reporter tags. We here present the construction of a set of nine E. coli-Mycobacterium shuttle plasmids, which offer a combination of three mycobacterial promoter systems (heat shock inducible-hsp60, tetracycline-, and acetamide-inducible) along with three polypeptide tags (Green Fluorescent Protein (GFP), Glutathione S-transferase (GST) and hexahistidine tag). These vectors offer the cloning of a target gene in all the nine given vectors in parallel, thus allowing the generation of recombinant plasmids that will express the target gene from different promoters with different tags. Here, while the hexa-histidine and GST tags can be used for protein purification and pull-down experiments, the GFP-tag can be used for protein localization within the cell. Additionally, the vectors also offer the choice of positioning of the reporter tag either at the N-terminus or at the C-terminus of the expressed protein, which is achieved by cloning of the gene at any of the two blunt-end restriction enzyme sites available in the vector. We believe that these plasmids will be extremely useful in the gene expression studies in mycobacteria by offering the choices of promoters and reporters. Our work also paves the way to developing more such plasmids with other tags and promoters that may find use in mycobacterial biology.
Journal of Biotechnology, 1994
A new positive selection vector, pGS23, based on the Lambda lysis cassette has been designed for efficient expression of homologous and heterologous genes in Escherichia coli. The plasmid permits controlled expression of a gene of interest under transcriptional control of the lac promoter with translation initiation of coding sequences directed by the phage T7 gene 10 ribosome binding site. The application of the vector system was tested for high level expression of the heterologous phbA gene of Alcaligenes eutrophus in E. coli.
Gene, 1986
Two new expression vectors have been constructed to take advantage of several useful properties of bacteriophage TCinfected Escherichia coli. These plasmids, pRDB8 and pRDB9, contain the promoter region and start codon of T4 gene 32, a contiguous multiple cloning site (MCS), and translation and transcription termination signals. DNA fragments inserted into the MCS are transcribed and translated at a high level in both uninfected and phage T6infected cells. Furthermore, the extreme stability of the hybrid mRNA after infection permits the specific biosynthetic labeling of the protein encoded by the cloned gene. In addition, the cloned gene product is stabilized, since the host-mediated degradation of foreign proteins is inhibited by phage infection. The properties of this expression system were demonstrated with the constant region of a rabbit immunoglobulin 1 light chain (C,) gene. Although proteolytic degradation of the C, fusion protein was rapid in uninfected cells, degradation was blocked in phage-infected cells and the protein accumulated in greater amounts.
Bacteriophage T4, a new vector for the expression of cloned genes
Gene, 1985
The amino-terminal portion of the T4 rIIB gene has been fused to the coding sequence of a truncated ZacZ gene from Escherichiu co& giving rise to a fusion protein with fi-galactosidase activity. The 3 192-bp rIIB-ZacZ gene fusion was transferred into phage T4, and enzymatically active protein was produced after phage infection. T4 may be a useful expression vector in special circumstances, in particular for proteins whose accumulation in E. cob is limited by sensitivity to proteases.
Infection and Immunity
The gene encoding the immunodominant 65-kilodalton antigen of Mycobacterium leprae was subcloned from a lambda gtll clone into the high-copy-number plasmid pUC8. Escherichia coli containing these recombinants produced large amounts of the antigen, which was purified by polyacrylamide gel electrophoresis in the presence of urea. The ability of E. coli to recognize the mycobacterial promoter was confirmed by constructing additional clones in which the gene is flanked by transcriptional terminators from phage fd. A similar approach was used to demonstrate the expression of this gene in Streptomyces lividans. Mice immunized with killed M. leprae showed cell-mediated immune reactivity to the purified 65-kilodalton protein which stimulated both in vitro lymphoproliferative and in vivo delayed-type hypersensitivity responses.
Construction of pMH, a Convenient Escherichia coli Protein Expression Vector
Molecular Biology, 2004
A new convenient vector, pMH, was designed for protein expression in Escherichia coli. The vector provides for effective inducible transcription of cloned sequences with T7 RNA polymerase and contains a polylinker harboring ten sites for the most common restriction enzymes, making it possible to clone a broad range of sequences. A region coding for the N-terminal (c-myc) 3-(His) 6 tag allows easy affinity purification of the recombinant protein and its detection with antibodies specific to the tag epitopes. The utility of pMH was demonstrated by successful expression of Drosophila melanogaster Chriz (CG10712) and its deletion derivatives in E. coli and purification of the recombinant proteins with a yield of about 10 mg per liter culture.
Cloning andAssessment ofMycobacterial Promoters by Using a Plasmid Shuttle Vector
1993
We have constructed a promoter selection vector for mycobacteria to analyze the sequences involved in mycobacterial transcriptional regulation. The vector pSD7 contains extrachromosomal origins of replication from Escherichia coli as well as from Mycobacterium fortuitum and a kanamycin resistance gene for positive selection in mycobacteria. The promoterless chloramphenicol acetyltransferase (CAT) reporter gene has been used to detect mycobacterial promoter elements in a homologous environment and to quantify their relative strengths. Using pSD7, we have isolated 125 promoter clones from the slowly growing pathogen Mycobacterium tuberculosis H37Rv and 350 clones from the fast-growing saprophyte Mycobacterium smegmatis. The promoters exhibited a wide range of strengths, as indicated by their corresponding CAT reporter activities (5 to 2,500 nmol/min/mg of protein). However, while most of the M. smegmatis promoters supported relatively higher CAT activities ranging from 100 to 2,500 nmol/min/mg of protein, a majority of those from M. tuberculosis supported CAT activities ranging from 5 to only about 100 nmol/min/mg of protein. Our results indicate that stronger promoters occur less frequently in the case of M. tuberculosis compared with M. smegmatis. To assess the extent of divergence of mycobacterial promoters vis-a-vis those of E. coli, the CAT activities supported by the promoters in E. coli were measured and compared with their corresponding activities in mycobacteria. Most of the mycobacterial promoter elements functioned poorly in E. coli. The homologous selection system that we have developed has thus enabled the identification of mycobacterial promoters that apparently function