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Biomedical subjects

P Balbás

Publications and source records attributed to P Balbás.

12 recordsLinked to original sources
PubMed2001

Chromosomal editing in Escherichia coli. Vectors for DNA integration and excision.

Chromosomal editing constitutes the direct and specific modification of the genetic information present in the chromosome. In the bacterium Escherichia coli, strategies were originally developed for the production of specific proteins, the genotypic improvement of strains, and the analysis of regulation of gene expression. However, with the emerging field of metabolic engineering and genomics, efficient means of targeting specific genetic mutations into the chromosome are most useful. In this review, a summary of the systems currently available to generate insertions and deletions in the chromosome of E. coli are presented, as well as the current knowledge about the genetic mechanisms responsible for these processes.

Animals↗
PubMed2001

Understanding the art of producing protein and nonprotein molecules in Escherichia coli.

The high-level production of functional proteins in E. coli is a very extense field of research in biotechnology. A number of important aspects to be considered in the initial design of an expression system and their interplay, were clear years ago. However, in recent times, strategies that go beyond transcription, translation, stability, vector, and strain choice, have been developed; so now expression of active peptides can be viewed as a more integrated process. Coexpression of protein subunits, foldases and chaperones, protein folding, location and purification schemes, metabolic engineering of the cell's central metabolism, and in vitro refolding strategies, are some of the novelties that are now available to aid in the success of an efficient expression system for active heterologous proteins. This review presents a compilation of the basic issues that influence the success in the production of protein and nonprotein products in Escherichia coli, as well as some general strategies designed to facilitate downstream process operations and improve biosynthesis yields.

Escherichia coli↗
PubMed1996

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome.

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211-213]. A family of related plasmids containing different antibiotic-resistance markers (CmR or GmR or KmR) and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.

Base Sequence↗
PubMed1994

Production in Escherichia coli of a rat chimeric proinsulin polypeptide carrying human A and B chains and its preparative chromatography.

A pseudohuman proinsulin coding DNA sequence (MMRPI) carrying human A and B chains, was constructed via directed mutagenesis of a previously modified rat proinsulin cDNA (MRPI) and expressed as a tryptophan (Trp)LE-proinsulin fusion protein in Escherichia coli W3110. Expression of the hybrid gene was achieved by depletion of tryptophan from the medium. The heterologous fusion protein, accumulated as insoluble inclusion bodies within the cell, was obtained by differential centrifugation and then solubilized using formic acid. At the junction of the two peptides, a methionine residue allowed proinsulin to be released from the carrier protein by cyanogen bromide treatment. The sulfonated form of this proinsulin polypeptide was easily purified, at a preparative level, using ion exchange chromatography.

Amino Acid Sequence↗
PubMed1994

Antisense overlapping open reading frames in genes from bacteria to humans.

Long Open Reading Frames (ORFs) in antisense DNA strands have been reported in the literature as being rare events. However, an extensive analysis of the GenBank database revealed that a substantial number of genes from several species contain an in-phase ORF in the antisense strand, that overlaps entirely the coding sequence of the sense strand, or even extends beyond. The findings described in this paper show that this is a frequent, non-random phenomenon, which is primarily dependent on codon usage, and to a lesser extent on gene size and GC content. Examination of the sequence database for several prokaryotic and eukaryotic organisms, demonstrates that coding sequences with in-phase, 100% overlapping antisense ORFs are present in every genome studied so far.

Amino Acid Sequence↗
PubMed1993

Plasmid pBRINT: a vector for chromosomal insertion of cloned DNA.

Plasmid pBRINT is a pBR322 derivative [Bolivar et al., Gene 2 (1977) 95-113; Balbás et al., Gene 50 (1986) 3-40] that allows the insertion and replacement of DNA sequences into the Escherichia coli chromosome by homologous recombination. This method uses the inability of E. coli strain ATCC47002 (JC7623) to replicate covalently closed circular (ccc) pBR322-derived plasmids, and the convenience of XGal+IPTG screening for recombinants. The vector also contains suitable selection markers (Ap and Cm), as well as a multiple cloning site (MCS) derived from the pUC vectors [Yanisch-Perron et al., Gene 33 (1985) 103-119] to facilitate cloning. A simple PCR scheme was developed to scan for DNA insertions into the bacterial chromosome. Once introduced into the chromosome, the inserted DNA sequences can be transferred to other strains by bacteriophage P1-mediated transduction.

Base Sequence↗
PubMed1992

Carbon regulation and the role in nature of the Escherichia coli penicillin acylase (pac) gene.

Quantitative analysis of specific pac mRNA and a lacZ fusion to the 5'-terminal region of the pac gene demonstrated that both phenylacetic acid induction and catabolite repression by glucose are involved, at the transcriptional level, in the regulation of the pac gene. The studies presented here suggest that this regulation is also present in Escherichia coli transformed strains in which the pac gene was not originally present. Analysis of the nucleotide sequence of the 5'-terminal region of this gene, with a statistical algorithm, confirms that the putative promoter previously proposed by our group is the most feasible within this region. We demonstrate that penicillin acylase activity can confer on E. coli the ability to use penicillin G as a metabolic substrate, by detaching the phenylacetic group which can be used as a carbon source. Based on these data, the regulation properties of the pac gene studied in this work, and the specificity profile of the penicillin acylase enzyme we suggest a role for it in E. coli as a scavenger enzyme for phenylacetylated compounds.

Base Sequence↗
PubMed1991

The role of penicillin amidases in nature and in industry.

Penicillin amidase (PA) is the enzyme used commercially for the production of semisynthetic penicillins. During the past decade, a detailed picture of the structure and regulation of the gene encoding this enzyme has emerged, revealing a variety of interesting features that are unique among microorganisms. Clues to the biological role of this enzyme have been provided, as well as new strategies for the commercial production and utilization of PA.

Escherichia coli↗
PubMed1986

Plasmid vector pBR322 and its special-purpose derivatives--a review.

The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichia coli. This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence. The widespread use of pBR322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmid's effectiveness as a cloning vector: plasmid instability in the absence of selection and, the lack of a direct selection scheme for recombinant DNA molecules. Several vectors based on pBR322 have been constructed to overcome these limitations and to extend the vector's versatility to accommodate special cloning purposes. The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322.

Base Sequence↗
PubMedDate not supplied

New insights on the comma-less theory.

The comma-less hypothesis represents a theoretical effort to describe one of the steps in the early evolution of the translation apparatus. This hypothesis emphasizes the advantages that a RNY coding pattern would have provided in a primitive RNA adaptor-catalyst system. This theory has been debated for years, both in conceptual and statistical terms, and no consensus about its validity has been ascertained. In this work, a statistical model refuting this theory was reconsidered. This new approach eliminates the bias due to the absence of stop codons in the open reading frame, and to the amino acid composition of bacterial genes. The results obtained support the biological significance of the RNY coding pattern.

Amino Acids↗