Biomedical subjects

Danqing Li

Publications and source records attributed to Danqing Li.

3 recordsLinked to original sources

PubMed2006

Serum-free suspension large-scale transient transfection of CHO cells in WAVE bioreactors.

Here, we report the development of a large-scale transient expression platform utilizing Chinese hamster ovary (CHO) cells. The majority of recombinant proteins and antibodies that are produced for preclinical models and clinical trials are expressed in stably transfected CHO cells. A protocol for transient transfection of CHO cells that is rapid, reproducible, and cost-effective would therefore streamline the process from research to development and help avoid any potential host species induced variation in the molecule of interest. CHO cells were adapted to grow in serum-free suspension conditions in spinner flask cultures in a proprietary in-house developed growth medium. In developing this transient transfection protocol, the parameters optimized included the transfection reagent of choice, the cell density at the time of transfection, the plasmid DNA concentration, and the transfection reagent concentration. Using this optimized protocol, we have expressed recombinant proteins, including antibodies, at an expression level of up to 9.4 mg/L. We also report transient transfections from 500 mL working volume (w.v.) up to 20 L w.v. in a WAVE bioreactor. Using this optimized protocol, it is possible to rapidly (within 10 d) produce up to 100 mg of recombinant protein for further study.

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PubMed2002

A new green fluorescent protein construct for localizing and quantifying peptide release.

Green fluorescent protein (GFP)-tagged secretory proteins recently have been used for studying packaging of peptides, secretory vesicle dynamics, and regulation of peptide release. In cells in which release occurs from sites with an abundance of vesicles, it is difficult to resolve the exact location of individual exocytotic events with standard wide-field epifluorescence microscopy. Furthermore, current GFP constructs are not well suited for real-time measurement of peptide release from large numbers of cells. Here, we describe a new pH-sensitive construct that is designed for localizing and quantifying release of neuropeptides and peptide hormones. Specifically, the yellow GFP variant called Topaz was fused to proAtrial natriuretic peptide (proANP, also called proAtrial natriuretic factor). The fluorescence of this fusion protein is low in normally acidic secretory vesicles but increases approximately 10-fold upon neutralization. Furthermore, it is released upon depolarization of PC12 cells. Finally, individual release events can be detected as brief localized flashes of fluorescence. ProANF-Tpz should prove useful for studying single release events by wide-field epifluorescence microscopy and for fluorometer-based real-time peptide release measurements from high numbers of cells.

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PubMed2002

Lack of sumatriptan-induced aortic contraction or relaxation: 5-HT1B receptor protein detected in endothelium and smooth muscle of vasa vasorum but not aorta.

Since 5-HT1B receptor mRNA was reported in rat aorta, and 5-HT1B receptor activation has been linked to vascular contraction, we explored sumatriptan-induced contractility and immunohistochemical density of 5-HT1B receptor protein in rat aorta. Sumatriptan (up to 10(-4) M), a 5-HT1B/1D receptor agonist, did not contract the endothelial intact or denuded rat aorta, even in the presence of L-NAME or after induction of modest tone with PGF2 alpha (10(-6) M). Sumatriptan also did not relax aortic preparations precontract with PGF2 alpha (3 x 10(-6) M) or phenylephrine (3 x 10(-7) M). Using a polyclonal antibody directed against the 5-HT1B receptor, minimal 5-HT1B receptor protein was detected in either the endothelium or smooth muscle of the rat aorta. However, dense 5-HT1B receptor protein was found in the vascular smooth muscle of the vasa vasorum supplying the aorta. Thus, the 5-HT1B receptor mRNA detected in tissue extracts of the rat aorta most likely reflects 5-HT1B receptor expression in the arterioles of the vasa vasorum. These studies support the link between the 5-HT1B receptor and vascular contraction in that the aorta with low density of 5-HT1B receptors lacked responses to sumatriptan, an agonist thought to contract blood vessels via 5-HT1B/1D receptors. Furthermore, caution must be observed when using 5-HT1B receptor mRNA to suggest receptor protein in tissues since this RT-PCR product may be derived predominantly from small blood vessels.

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