High Copy Number Vector Research Articles

Phospholipase A2 engineering: design, synthesis, and expression of a gene for bovine (pro)phospholipase A2.

A gene coding for the (pro)phospholipase A2 (PLA2) from bovine pancreas has been designed, synthesized, and expressed in Escherichia coli. The gene was designed with a variety of restriction sites that will facilitate future mutagenesis studies. Codons occurring frequently in prokaryotic systems were chosen whenever possible. The total gene spans 404 base pairs and was divided into 33 oligonucleotides. The gene was constructed in two halves of 224 and 180 base pairs from the oligonucleotides by the shotgun ligation technique using pBSM13- as the cloning vehicle. The two fragments were then ligated and cloned into pBSM13- to complete the gene. The (pro)PLA2 gene was then verified by restriction site mapping and dideoxy sequencing. The gene was expressed to high levels from a high copy number vector, designated as pJPN, derived from the E. coli secretion vector pIN-III-ompA3. Although the protein failed to be excreted and was in the form of insoluble inclusion body, active PLA2 could be obtained by renaturation of the inclusion body pellet followed by tryptic activation, which removes the signal sequence and the pro-peptide of proPLA2. The PLA2 thus obtained reacted with the antisera raised against the natural PLA2 purified from bovine pancreas, and the specific activity of the expressed PLA2 was identical to that of the natural PLA2. The shotgun ligation and synthetic gene approaches are simple and inexpensive and can be adapted to express most of the enzymes in the phospholipase A2 family.

Plasmid pMTL153: a high copy number version of pAT153 and its use to obtain high expression of the Pseudomonas carboxypeptidase G2 gene

High level expression (27% of the cells soluble protein) of the potentially important therapeutic enzyme carboxypeptidase G2 (CPG2) has been achieved by placing the encoding gene under the transcriptional control of the E. coli lac promoter and using a high copy number version of the plasmid pAT153. The high copy number vector, pMTL153, was constructed by replacing the replication region of pAT153 with the equivalent region from pUC8. The resultant 3-fold increase in copy number was due to a previously unreported nucleotide substitution in the RNA I/RNA II region of pUC plasmids. Evidence is also presented that production of β-lactamase from the vector encoded bla gene dramatically reduces the levels of the periplasmically located CPG2 product. Substitution of the vector bla gene with tet (non-periplasmic gene product) resulted in a significant increase in the levels of CPG2 obtained. This finding may be relevant to the expression of other secreted heterologous proteins

Three nuclear genes suppress a yeast mitochondrial splice defect when present in high copy number

A gene bank of a yeast wild type DNA in the high copy number vector YEp13 was screened for recombinant plasmids which suppress the mitochondrial RNA splice defect exerted by mutant M1301, a -1 bp deletion in the first intron of the mitochondrial COB gene (bI1). A total of 17 recombinant plasmids with similar suppressor activity were found. Restriction mapping and cross-hybridization of the inserts revealed that these 17 plasmids contain three different inserts, all lacking any extended sequence homology. Each of the inserts, when present in high copy number, has a similar suppressor activity: high in the presence of mutation M1301 in bI1, a group II intron, and low but significant with the presence of few mutants in bI2 and bI3 of the COB gene, both of which are group I introns.

Cloning and characterization of the gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae.

The LPD1 gene of S. cerevisiae, which encodes lipoamide dehydrogenase (EC 1.8.1.4), has been cloned and characterized. The LPD1 gene is present as a single copy in the yeast genome and is transcribed to give a polyadenylated mRNA species of approximately 2.0 kb. The synthesis of lipoamide dehydrogenase in yeast is subject to carbon catabolite repression since both the level of the LPD1 transcript and the accumulation of the lipoamide dehydrogenase subunit polypeptide were greatly reduced in wild-type cells grown on glucose compared to those grown on a variety of non-fermentable carbon sources. Strains defective in LPD1 but transformed with the LPD1 gene on a high copy number vector exhibited elevated levels of the LPD1 transcript as well as increased lipoamide dehydrogenase activity when grown on glycerol. Immunoblotting experiments confirmed that such transformants over-expressed lipoamide dehydrogenase protein. Transcription from the LPD1 sequence on plasmid pGP1 still appeared to be subject to some catabolite repression despite the presence of multiple copies of the plasmid in the cell.

Effect of multiple copies of rpoBC on the rate of RNA synthesis in Escherichia coli.

SummaryThe cloning of therpoBandrpoCgenes in a high copy number vector inE. coliincreased the amount of the encoded gene products, the β and β′ subunits of RNA polymerase. However, this unexpectedly caused a 30–50% decrease in RNA synthetic activity which alternatively induced a reduction of growth rate and enlargement of cell size, and decreased the DNA replication time. The results can be explained by autogenous regulation of the RNA polymerase genes by the ββ′ subunits. A relation between the decrease in number of transcription units and the observed higher rate of movement of DNA replication forks is discussed.

Secretion cloning vectors for guiding the localization of proteins in vivo.

Cloning of genes into high copy number vectors has supplied biochemists with a powerful method for the production and purification of large amounts of biochemically important macromolecules. Judicious choice of promoter sequences has produced vectors capable of loading cells with 50% of the total cellular protein as the desired gene product, making purification a trivial chore. However, this approach is limited to proteins whose activity is not detrimental to protein production, and to proteins not sensitive to cytoplasmic proteases (see below). Recognition of this problem has stimulated the development of several cloning vectors designed to guide a cloned gene product out of the cytoplasm of the bacterial cell. These vectors produce a hybrid protein in which the cloned gene product is fused with a signal peptide, a structural element necessary for protein translocation. In this review, we will describe the advantages and limitations of some currently available secretion cloning vectors, and will discuss the use of these tools to produce large amounts of protein and to determine the information required to direct proteins to have various compartments of the cell.

Isolation of the gene encoding adenylate cyclase in Saccharomyces cerevisiae.

By complementation of the cyr1-1 mutation in Saccharomyces cerevisiae, we have isolated yeast genomic DNA containing the structural gene that encodes the catalytic unit of adenylate cyclase (EC 4.6.1.1). The isolated DNA restored adenylate cyclase activity to cyr1-1 mutants and directed integration at the CYR1 locus. Wild-type strains transformed with CYR1 DNA on the high copy number vector YEp24 contained 4- to 6-fold more adenylate cyclase activity than strains carrying the plasmid with no insert. This result suggests that expression of the CYR1 gene product, rather than that of other polypeptide components of the adenylate cyclase system, limits total adenylate cyclase activity in S. cerevisiae. CYR1-containing plasmids also complemented the temperature-sensitive growth defect of the cell division cycle mutation cdc35-1, which confers a phenotype under restrictive conditions similar to that of cyr1-1 and maps to the same locus. Further, cdc35-1 cam mutants, which contain mutations that enable them to take up cAMP from the medium, grew at the restrictive temperature in the presence of exogenous cAMP. These observations support the view that CDC35 and CYR1 are allelic and confirm the hypothesis that cAMP synthesis is required for cells to pass through the "start" position of the cell division cycle.

Identification of plasmid (pKM101)-coded proteins involved in mutagenesis and UV resistance.

Mutagenesis of Escherichia coli by UV and many chemicals is not a passive process and requires the intervention of host functions. The isolation of chromosomal umuC mutants, which are nonmutable by many DNA-damaging agents and are slightly sensitive to them, has led to the model that the umuC gene product(s) functions in a pathway of ‘error-prone repair’1–3. UmuC mutants are suppressed by the introduction of the plasmid pKM1014; pKM101 was derived from the clinically isolated plasmid R46 by in vivo manipulations5. The muc (mutagenesis, UV and chemical) region of pKM101 is required for this suppression and we have suggested that it is an analogue of the chromosomal umuC gene(s)6. We have now subcloned the muc region onto the high-copy number vector pKB354 and identified two protein products by gel electrophoresis of maxicell preparations. Complementation analysis of muc insertion mutations has confirmed the existence of two genes, mucA and mucB, whose protein products are required for pKM101's effects on mutagenesis and resistance to killing. The molecular weights (MWs) of the mucA and mucB proteins are 16,000 and 45,000 respectively.