Cloning and expression of human haptoglobin subunits in Escherichia coli: Delineation of a major antioxidant domain

Lai, I-Hsiang; Tsai, Tsung I.; Lin, Hong; Lai, Wei Yen; Mao, Simon J.T.

doi:10.1016/j.pep.2006.09.012

Cited by 11 publications

(9 citation statements)

References 33 publications

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“…Molecular-mass standard containing 12 prestained proteins (3.5–260 kDa) was purchased from Invitrogen (Carlsbad, CA, USA). Western blot analysis was performed similar to that described previously [13]. …”

Section: Methodsmentioning

confidence: 99%

“…One of the major functions of Hp is to capture released hemoglobin during excessive hemolysis [12] and to scavenge the hemoglobin-induced free radicals during oxidative stress [16]. We have recently shown that Hp is an extremely potent antioxidant that directly prevents low-density-lipoproteins from Cu 2+ - and radical compound-induced oxidation [13, 30]. Transfection of Hp cDNA into Chinese hamster ovary cells protects them against oxidative stress [30].…”

Section: Introductionmentioning

confidence: 99%

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Neutrophils as one of the major haptoglobin sources in mastitis affected milk

Lai

Tsao²,

et al. 2008

Vet. Res.

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The antioxidant haptoglobin (Hp) is an acute-phase protein responsive to infectious and inflammatory diseases. Hp and somatic cell counts (SCC) are sharply elevated in bovine milk following intramammary administration of endotoxin or bacteria. However, the sources of milk Hp responsible for such increases are not fully understood. The purpose of this study was to define the source of milk Hp from dairy cows with naturally occurring mastitis. Quarter milk samples selected from 50 dairy cows were separated into four groups according to SCC as group A: < 100 (n = 19); B: 100–200 (n = 10); C: 201–500 (n = 10); and D: > 500 × 103 (n = 11) cells/mL. Our results reveal that milk Hp concentrations were correlated with SCC (r = 0.742; P < 0.01), and concentrations in group D were ~10-fold higher than in group A. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that the milk somatic cells from group D were not only capable of synthesizing Hp but could also markedly increase Hp mRNA expression. Western blot, immunocytochemistry, double confocal immunofluorescence, and Hp releasing experiments demonstrate that neutrophils were associated with the biosynthesis and release of Hp in milk. It further shows that Hp was significantly elevated in the epithelium of mammary gland tissue with mastitis and was also expressed in the cultured mammary epithelial cells. We propose that neutrophils and epithelial cells may play an essential role in elevating milk Hp in addition to previous suggestions that Hp may be derived from mammary tissues and circulation.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Neutrophils as one of the major haptoglobin sources in mastitis affected milk

Lai

Tsao²,

et al. 2008

Vet. Res.

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“…The first strand cDNA was synthesized with M-MLV reverse transcriptase at 37°C for 50 min. A gene fragment coding for porcine Hp was amplified by polymerase chain reaction (PCR) using proofreading DNA-polymerase and oligonucleotide primers as described previously [21]. The primer design was based on the published nucleotide sequence of porcine Hp (NM_214000).…”

Section: Methodsmentioning

confidence: 99%

Low Levels of Haptoglobin and Putative Amino Acid Sequence in Taiwanese Lanyu Miniature Pigs

Yueh

Wang

Lin

et al. 2008

The Journal of Veterinary Medical Science

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ABSTRACT. Porcine haptoglobin (Hp) is an acute phase protein. Its plasma level increases significantly during inflammation and infection. One of the main functions of Hp is to bind free hemoglobin (Hb) and inhibit its oxidative activity. In the present report, we studied the Hp phenotype of Taiwanese Lanyu miniature pigs (TLY minipigs; n=43) and found their Hp structure to be a homodimer (β-α-α-β) similar to human Hp 1-1. Interestingly, Western blot and high performance liquid chromatographic (HPLC) analysis showed that 25% of the TLY minipigs possessed low or no plasma Hp level (<0.05 mg/ml). The Hp cDNA of these TLY minipigs was then cloned, and the translated amino acid sequence was analyzed. No sequences were found to be deficient; they showed a 99.7% identity with domestic pigs (NP_999165). The mean overall Hp level of the TLY minipigs (0.21 ± 0.25 mg/ml; n=43) determined by enzyme-linked immunosorbent assay (ELISA) was markedly lower than that of domestic pigs (0.78 ± 0.45 mg/ml; p<0.001), while 25% of the TLY minipigs had an Hp level that was extremely low (<0.05 mg/ml). In addition, the initial recovery rate (first 40 min) in the circulation of infused fluorescein isothiocyanate (FITC)-Hb was significantly higher in the TLY minipigs with extremely low Hp levels than those with high levels. This data suggests that the low concentration of Hp-Hb complex is responsible for the higher recovery rate of Hb in the circulation. TLY minipigs have been used as an experimental model for cardiovascular diseases; whether they can be used as a model for inflammatory diseases, with Hp as a marker, remains a topic of interest. However, since the Hp level varies significantly among individual TLY minipigs, it is necessary to prescreen the Hp levels of the animals to minimize variation in the experimental baseline. The present study may provide a reference value for future use of the TLY minipig as an animal model for inflammation-associated diseases. KEY WORDS: amino acid sequence, evolution, haptoglobin concentration, hemoglobin, Lanyu minipig.

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“…In general, the protein samples were incubated at 70°C for 10 min in a NuPAGE LDS sample buffer (Invitrogen) before being loaded onto to the gel. The proteins were visualized by Coomassie blue staining or electrotransferred onto an Immobilon-P membrane (Millipore, Bedford, MA, USA) and processed for Western blot analysis as described previously [15]. In brief, electrotransferred and blocked membrane was incubated with a relevant primary antibody (rabbit antiserum against B. pseudomallei [1:1,000 dilution], mouse monoclonal antibody against His-tag [Invitrogen] [1:1,000 dilution], or a culture-confirmed melioidosis goat serum [1:100 dilution]), followed by washes with PBS containing 0.05% Tween 20 (PBS-T) and incubation with the relevant secondary antibody, goat anti-rabbit IgG peroxidase conjugate (KPL, Gaithersburg, MD, USA), goat anti-mouse IgG peroxidase conjugate (KPL), or rabbit anti-goat IgG peroxidase conjugate (KPL; 1:5,000 dilution).…”

Section: Sds-page and Western Blot Analysesmentioning

confidence: 99%

Development of an immunoassay using recombinant outer membrane protein A and flagellin for diagnosis of goats with melioidosis

Lee

Shih

et al. 2020

The Journal of Veterinary Medical Science

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Among domestic animals, melioidosis is one of the most common diseases reported in goat, sheep, and swine. To evaluate the specific antibodies in goats with melioidosis, we developed a serology test using recombinant outer membrane protein A (OmpA) and flagellin (FliC) of Burkholderia pseudomallei as antigens. DNA corresponding to each antigen was cloned into a pET32a vector and expressed in Escherichia coli. Essentially, the recombinant OmpA and FliC were expressed in a soluble form that could be isolated with 95% homogeneity. Both recombinants could be recognized by rabbit antibodies prepared against heat-inactivated B.

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Cloning and expression of human haptoglobin subunits in Escherichia coli: Delineation of a major antioxidant domain

Cited by 11 publications

References 33 publications

Neutrophils as one of the major haptoglobin sources in mastitis affected milk

Neutrophils as one of the major haptoglobin sources in mastitis affected milk

Low Levels of Haptoglobin and Putative Amino Acid Sequence in Taiwanese Lanyu Miniature Pigs

Development of an immunoassay using recombinant outer membrane protein A and flagellin for diagnosis of goats with melioidosis

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