Antibody-free method for protein detection on blots using enzyme fragment complementation.
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Peter Fung
Publications and source records attributed to Peter Fung.
Stability of infectious recombinant adeno-associated viral vector in gene delivery.
BACKGROUND: The aim of this study is to provide a basis for the design of appropriate protocols for the shipping and storage of rAAV vectors for experimental laboratory studies and clinical trials. MATERIAL/METHODS: rAAV stocks were generated by standard methods and then subjected to different environments. The transduction efficiency of viral vectors both in vitro and in vivo was determined by luciferase activity and immunohistochemistry. RESULTS: The virus stored at -80 degrees C remained completely stable and had high transduction efficiency. By contrast, the transduction efficiency of all other groups on 293 cells decreased continuously over time. The transduction efficiency of the -20 degrees C group remained relatively high for the first 5 days, but dropped sharply between days 5 and 7. The transduction efficiency for the 4 degrees C group dropped sharply on both days 1 and 7, and continued to decrease to 55% of maximum efficiency by the end of the first month. For both the room temperature (RT) and 37 degrees C groups, a sharp fall in efficiency was observed at day 1, and efficiency continued to decline throughout the experimental period. Data from the in vivo study also revealed that rAAV vector stored at -80 degrees C remained stable and retained its transduction efficiency. CONCLUSIONS: The virus stored at -80 degrees C remained completely stable and retained high transduction efficiency. The implications of these findings provide a basis for viral stock portioning and avoidance of freeze-thawing and storing at temperatures above -80 degrees C prior to clinical trials.
Animals↗
Homogeneous assays for cellular protein degradation using beta-galactosidase complementation: NF-kappaB/IkappaB pathway signaling.
Activation of cells by the tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) cytokines results in activation of the nuclear factor-kappaB (NF-kappaB) via proteasomal degradation of an associated IkappaB molecule. To monitor cellular IkappaB, the protein was recombinantly expressed as a fusion protein with a novel enzymatic tag, ProLabel (PL). ProLabel is a small 5.5-kDa sequence from the amino-terminal amino acids of beta-galactosidase, possesses a simple ribbon structure, and can be fused to many proteins via the amino or carboxyl terminus. Expression of this construct allows quantitative detection of the recombinant protein in crude lysates by using a method based on beta-galactosidase enzyme fragment complementation (EFC). Transient transfection of IkappaB-PL in HeLa cells generated an EFC signal that was highly correlated with a western analysis of the protein construct. ProLabel expressed alone in the cells did not show any EFC activity, due to rapid proteolytic degradation, indicating a very low background signal from the protein tag. TNF-alpha and IL-1 treatment induced a concentration-dependent degradation of IkappaB-PL, with potency values similar to those reported using other methods. IkappaBM-PL (mutant of IkappaB-PL), in contrast, did not undergo degradation for concentrations up to and including 10 ng/ml TNF-alpha or IL-1, demonstrating that degradation of IkappaB-PL was specific to the NF-kappaB pathway activation. TNF-alpha and IL-1 induced maximal IkappaB-PL degradation within 30 min of induction. This was reversed by several agents that ablate this pathway, including anti-TNF-alpha antibodies and the proteasome inhibitor, MG-132. The assay was amenable to HTS systems, with good precision and reproducibility. Z' values and coefficients of variance for IkappaB-PL degradation were 0.6 and <9%, respectively.
Dose-Response Relationship, Drug↗