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E Curiel-Quesada

Publications and source records attributed to E Curiel-Quesada.

7 recordsLinked to original sources
PubMed2001

Isolation of human scFv antibody fragments against ABO blood group antigens from a phage display library.

BACKGROUND AND OBJECTIVES: Phage display has proven very useful for the isolation of antibodies against a number of antigens. We used this technology to isolate scFv antibody fragments against A and B red blood cell antigens. MATERIALS AND METHODS: Phages from a phage display library were selected using unmodified red blood cells as a source of antigen. Bound phages were absorbed onto cells lacking the antigen of interest and used to infect Escherichia coli cells. Phages were rescued and assayed for specificity by enzyme-linked immunosorbent assay (ELISA). RESULTS: After several rounds of panning and subtraction on red blood cells, one anti-A and one anti-B human scFv antibody fragments were isolated. CONCLUSION: Isolation of anti-A and anti-B scFv antibody fragments on whole cells is an alternative method of obtaining antibodies against native cell-surface antigens.

ABO Blood-Group System↗
PubMed2001

Construction of a multiframe vector to express coding sequences in Escherichia coli.

Cloning of foreign DNA fragments for coding sequence analysis in Escherichia coli usually involves sets of three vectors. To simplify this, we constructed an expression vector named pMFV7 containing three ATG codons in different frames downstream of a Shine-Dalgarno sequence, assuming that the ribosome can use any of the three start codons in an alternative manner. Translation beginning at either of the start codons would drive the expression of any coding fragment cloned downstream. To test the feasibility of this proposal, we cloned DNA fragments of the lacZ gene in each of the possible reading frames downstream from pMFV7 start codons. Sequence analysis of the N-terminus regions around the fusion sites indicates that ribosomes indeed initiate translation at each of the three initiation codons. In one case, levels of beta-galactosidase activity depended largely on the N-terminus of the translation products. We conclude that pMFV7 may be useful for expressing coding sequences regardless of their reading frame.

Cloning, Molecular↗
PubMed2000

[Current knowledge on the strain typing of the pathogenic fungus Histoplasma capsulatum var. capsulatum: a review of the findings].

The classification of microbial strains is currently based on different typing methods, which must meet certain criteria in order to be widely used. Phenotypic and genotypic methods are being employed in the epidemiology of several fungal diseases. However, some problems associated to the phenotypic methods have fostered genotyping procedures, from DNA polymorphic diversity to gene sequencing studies, all aiming to differentiate and to relate fungal isolates or strains. Through these studies, it is possible to identify outbreaks, to detect nosocomial infection transmission, and to determine the source of infection, as well as to recognize virulent isolates. This paper is aimed at analyzing the methods recently used to type Histoplasma capsulatum, causative agent of the systemic mycosis known as histoplasmosis, in order to recommend those that yield reproducible and accurate results.

English Abstract↗
PubMed1998

Cloning and sequencing of the gene that codes for the Klebsiella pneumoniae GroEL-like protein associated with ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease of the sacroiliac joints and vertebral column of unknown aetiology, but strongly related to the presence of the HLA-B27 antigen. The participation of bacterial infections as triggering factors have also been suggested. We have associated the 60 kDa heat shock protein of Klebsiella pneumoniae (HSP60Kp) with AS since we have previously demonstrated that most of the patients have IgG antibodies and active T cells that recognize preferentially this protein, but we have not yet identified the epitopes involved in the recognition. In order to know the amino acid sequence of HSP60Kp, and to be able to analyse in the future the relevant epitopes; we amplified by PCR and cloned the gene coding for this protein into the SmaI site of pUC19. The nucleotide sequence of the gene was obtained by the Sanger method using both manual and automatic techniques. Amino acid sequence of the HSP60Kp was deduced by translating the nucleotide sequence of the gene. The antigenic analysis of this sequence was compared to the antigenic analysis of the reported sequences of Escherichia coli GroEL and Yersinia enterocolitica HSP60. Using a software to predict HLA class I motifs, the nonapeptide (KRGIDKAVL) residues 117-125 of HSP60Kp showed a much higher affinity for HLA-B27 than the similar nonapeptide of E. coli GroEL and Y. enterocolitica HSP60. The only difference between the three peptides was in position nine. This finding could explain the association of AS only with the HSP60 of Klebsiella pneumoniae. On the other hand, hydrophilicity analysis, which indicates B cell epitopes, showed three similar strongly antigenic regions in the three proteins.

Amino Acid Sequence↗
PubMed1984

Cloning of a new low-molecular-weight spore-specific protein gene from Bacillus megaterium.

Three EcoRI fragments of Bacillus megaterium DNA hybridized only under nonrestrictive conditions on Southern blots to a probe containing the previously cloned gene for protein C, a small, acid-soluble spore protein (SASP) from B. megaterium. All three fragments were cloned in Escherichia coli cells in plasmid pBR325, and after being transferred to an E. coli expression vector, one of the fragments (C-3) directed the synthesis of a new small, acid-soluble spore protein (termed C-3) immunologically related to protein C. As previously observed with the protein C gene, protein C-3 gene expression in E. coli required an external promoter and suppression of termination of transcription. Protein C-3 was purified from induced E. coli cells, and its immunological properties, electrophoretic mobility, amino acid composition, and amino-terminal sequence were determined. These data indicated that protein C-3 was related, but not identical, to either protein C or the closely related protein A--two of the major small, acid-soluble spore proteins of B. megaterium. Detailed examination of acid extracts of B. megaterium spores showed that they contained a minor protein which comigrated with C-3 on acrylamide gel electrophoresis at low pH and reacted immunologically like C-3.

Antigens, Bacterial↗
PubMed1983

Cloning of the gene for C protein, a low molecular weight spore-specific protein from Bacillus megaterium.

The structural gene for C protein, a low molecular weight spore-specific protein from Bacillus megaterium, has been cloned in Escherichia coli. Expression of the C-protein gene in E. coli requires an external transcription promoter and prevention of termination of transcription prior to transcription of all or part of the sequence coding for the C protein. The gene for the C protein is within a 5-kilobase DNA fragment, but this fragment does not code for either of the other two major low molecular weight spore proteins, suggesting that the structural genes for these proteins are not tightly linked.

Bacillus megaterium↗