David Shiuan

Determination of biotin concentration by a competitive enzyme-linked immunosorbent assay (ELISA) method

A method based on competitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of biotin concentrations which takes advantage of the extraordinarily high affinity between biotin and avidin. The biotin assay consisted of two steps, (i) a competition reaction between excess streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and solutions of known biotin concentrations or sample solutions and (ii) the measurement of the residual activities of the free form streptavidin-HRP which were correlated with the initial biotin concentrations. The procedure was modified by including an extra step of antibody-antigen interaction to assay biotin concentration unambiguously in more complex media. The entire assay was completed within 6 with sensitivities of approximately 1 pg/ml for biotin in a simple aqueous medium and 5 pg/ml in complex media. The method offers significant advantages in time, sensitivity and simplicity for determinations of biotin concentrations in various solutions.

Molecular cloning and analysis of a HSP (heat shock protein)-like 42 kDa antigen gene of Mycoplasma hyopneumoniae.

The recombinant clone expressing the 42 kDa protein (P42) of Mycoplasma hyopneumoniae in Escherichia coli was analyzed. The 4.4 kb HindIII‐XmaI DNA fragment expressing the p42 gene product encodes three ORFs: p42 and pl6 in the forwarding strand, p24 in the reverse strand. Sequence comparisons revealed that p42 could be part of a p65 gene, and has 62% identities with Mycoplasma genitalium HSP70 gene and 56% identities with Bacillus subtilis dnaK gene; p16 and p24 genes share 73% and 47% identities with Erysipelothrix rhusiopathiae dnaJ gene and Pseudomonas fluorescens uvrC gene, respectively. Further analysis demonstrated that P42 is indeed a heat shock protein and the monospecific antibodies against P42 can block the growth of Mycoplasma hyopneumoniae.

Rapid puritification of antiserum against Mycoplasma hyopneumoniae by an efficient absorption method

A simple and efficient method for the removal of unwanted cross-reactive antibodies has been developed. The antiserum purification method was based on treatment of the antiserum with both sonicated extracts and boiling extracts of the Escherichia coli host cells used in immunoscreening the lambda EMBL3 library. We have demonstrated unambiguously that through this simple treatment, the rabbit anti-Mycoplasma hyopneumoniae antiserum can be effectively purified so that the amount of antibodies cross-reacted with Escherichia coli lysate proteins is drastically reduced. Compared with the traditional absorption methods, which require the chemical coupling of an absorbing agent to an insoluble support, and affinity purification methods, which have harsh denaturing condition, this method should greatly facilitate a successful immunoscreening experiment.

Competitive enzyme-linked immunosorbent assay for biotin.

Publisher Summary This chapter proposes a competitive enzyme-linked immunosorbent assay (ELISA) method for the rapid and sensitive determination of biotin concentrations. This method is based on the measurement of residual horseradish peroxidase [conjugated to streptavidin, streptavidin–horseradish peroxidase (HRP)] activities after streptavidin–HRP has reacted with biotin in sample solutions. The detection limit is 1 pg/ml in simple aqueous media and 5 pg/ml in bacterial growth media. The strategy of the competitive enzyme-linked immunosorbent assay (ELISA) method for the determination of biotin concentration is based on the competition between streptavidin-conjugated horseradish peroxidase (streptavidin–HRP, in excess amount) in solutions of variable biotin concentrations. The residual free form of streptavidin–HRP is immobilized with biotinylated goat anti-rabbit immunoglobulin (IgG) precoated on the wells of microtiter plates, and finally, the peroxidase activities are assayed and correlated with the biotin concentrations. When more biotin molecules are present initially in the competition reactions, less free streptavidin–HRP would be available to react with the biotinylated IgG. Biotin concentrations could be estimated from the calibration curves relating standard biotin concentrations and the horseradish peroxidase activities.

A simple method for DNaseI footprinting analysis.

DNaseI footprinting technique has been a very useful method in gaining direct and immediate information about the location of a protein binding site in the DNA sequence. In this report, we present a method that overcomes many of the inconveniences of previous methods. This method was based on the combination of PCR technique and the dideoxy DNA sequencing reaction. It avoids the need of the secondary restriction sites that any candidate DNA sequence for DNaseI footprinting analysis can be prepared efficiently as long as its flanking sequences are known. Replacing the difficult Maxam-Gilbert DNA sequencing with the dideoxy-mediated DNA sequencing method makes the footprinting experiments even safer and easier.

Isolation and characterization of the Erwinia herbicola bio operon and the sequences of the bioA and bioB genes

The biotin operon of Erwinia herbicola was cloned and characterized. The operon consists of five genes arranged in the order, bioABFCD. The operon is negatively regulated via the interaction of a proposed biotin repressor with an operator sequence that lies between the bioA and bioB genes. The nucleotide sequences of bioA (7,8-diaminopelargonic acid transferase), bioB (biotin synthetase) and the regulatory region were determined and analyzed. The deduced amino acid sequences of bioA and bioB are also aligned with currently available homologs to obtain the UPGMA (unweighted pair group method with arithmetic mean) evolutionary tree.

Bioassay of biotin concentration with a Escherichia coli bio deletion mutant.

Biotin concentration was determined unequivocally with the E. coli bio mutant. The results demonstrate that this simple and efficient method can determine biotin concentration in the range of 10 pg to 50 ng/ml. The present method can also clearly distinguish biotin from its precursor and analog, dethiobiotin.

Transcriptional analysis of the Escherichia coli bio operon.

Using primer extension, two in vivo transcription start points (tsp) were identified for rightward transcription of the Escherichia coli biotin operon, at nucleotides (nt) +20 and +29. The strongest leftward transcript begins at +9, with a tenfold less abundant transcript starting at +3. The activity of segments cloned into promoter probe vectors locates the major leftward promoter between nt +1 and +105, as expected for the +9 tsp. Although the activity of a chromosomal operator is reduced about 300-fold by point mutation in either arm of the palindrome extending from nt -20 to +20, either a half-operator segment or a full operator bearing the same point mutation in one arm is substantially repressed when cloned into pKB2000, as though cellular location strongly affects the operators affinity for the repressor.

Molecular cloning and nucleotide sequencing of bioF (7-keto-8-amino pelargonic acid synthetase), bioC and bioD (dethiobiotin synthetase) genes of Erwinia herbicola.

The biotin operon of Erwinia herbicola Eho 10 was cloned and characterized by complementation of E. coli biotin mutants. The operon was found to contain five genes arranged in the order, bioABFCD. The nucleotide sequences of bioF (7‐keto‐8‐aminopelargonic acid synthetase), bioC and bioD (dethiobiotin synthetase) were determined and analyzed. The nucleotide sequences and deduced amino acid sequences of bioFCD were compared with the corresponding sequences from Escherichia coli, Bacillus sphaericus, Serratia marcescens and Brevibacterium flavum.

Construction and Characterization of A Biotin-Regulated Gene Expression System in Escherichia coli

An autoregulated gene expression system inEscherichia coli was designed such that the cloned genes on the vector were not expressed until biotin was depleted during cell growth. The expression vectors were constructed by assembling the DNA fragments containing the regulatory region of theE. coli biotin operon (bio operon), the universal ribosome-binding site (RBS) and the strong transcription terminator rrnBT1T2. The promoter region was further modified by site-directed mutagenesis to create promoters of varied strength. The feasibility of this system was examined inE. coli strain R901 (withbio operon deleted) using various marker genes, including theE. coli birA gene, T7 RNA polymerase gene and yellowfin-porgy growth-hormone gene. The results demonstrated that the induction of marker-gene expression can be triggered as the biotin concentration drops to a threshold value of approximately 2 ng/mL by metabolic utilization.