Browsing by Author "Cheng, Chiu-Min"

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A regularly spaced and self-revealing protein ladder for anti-tag Western blot analysis
(2012-12-01)
Kao, Chien-Han
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Cheng, Chiu-Min
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Chuang, Kuo-Hsiang
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Chuang, Chih-Hung
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Tzou, Shey-Cherng
We designed a protein ladder (hereafter referred to as "Mega-tag") that contains 14 of the most commonly used epitope tags fused to molecular weight markers. The Mega-tag ladder can be simultaneously visualized when anti-tag antibodies are used to detect epitope-tagged recombinant proteins in Western blots. The logarithm of molecular weights and relative mobility of the Mega-tag protein ladder are highly correlated (R-2 = 0.997 +/- 0.00232), indicating that the dye-free Mega-tag protein ladder is accurate. It can also serve as a positive control for anti-epitope tag immunoblots. The Mega-tag protein ladder should provide a convenient and precise tool for Western blot analysis. (c) 2012 Elsevier Inc. All rights reserved.
Development of a highly sensitive enzyme-linked immunosorbent assay (ELISA) through use of polyprotein G-expressing cell-based microplates
(2018-12-14)
Chen, Yi-Jou
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Chen, Michael
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Hsieh, Yuan-Chin
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Su, Yu-Cheng
;
Wang, Chang-Hung
The sensitivity of traditional enzyme-linked immunosorbent assays (ELISAs) is limited by the low binding avidity and heterogeneous orientation of capture antibodies coated on polystyrene-based microplates. Here, we developed a highly sensitive ELISA strategy by fixing poly-protein G-expressing cells on microplates to improve the coating amount and displayed orientation of capture antibodies. One or eight repeated fragment crystallisable (Fc) binding domains of protein G are stably expressed on the surface of BALB/c 3T3 cells (termed 1pG cells or 8pG cells), which then act as highly antibody-trapping microparticles. The 8pG cells showed higher antibody-trapping ability than the 1pG cells did. The antibody-coating amount of the 8pG cell-based microplates was 1.5-23 times and 1.2-6.8 times higher than that of traditional polystyrene-based and commercial protein G-based microplates, respectively. The 8pG cell-based microplates were then applied to an anti-IFN-alpha sandwich ELISA and an anti-CTLA4 competitive ELISA, respectively, and dramatically enhanced their detection sensitivity. Importantly, direct coating unpurified capture antibody produced by mammalian cells did not impair the antigen-capturing function of 8pG cell-based microplates. The 8pG cell-based microplates exhibited a significant improvement in antibody-coating amount and preserved the homogeneous orientation of capture antibodies, making them a potential replacement for traditional microplates in various formats of ELISAs.
Functional Production of a Soluble and Secreted Single-Chain Antibody by a Bacterial Secretion System
(2014-05-13)
Cheng, Chiu-Min
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Tzou, Shey-Cherng
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Zhuang, Ya-Han
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Huang, Chien-Chiao
;
Kao, Chien-Han
Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (alpha EGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate alpha EGFR-osmY, osmY-alpha EGFR, and pelB-alpha EGFR (control), respectively. In comparison with the control, both the osmY-fused alpha EGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble alpha EGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-alpha EGFR. In addition, the antigenbinding activity of both the osmY-fused alpha EGFRs was 2-fold higher than that of the refolded pelB-alpha EGFR from inclusion bodies. Similar results were observed with alpha TAG72-osmY and alpha Her2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost alpha EGFRprotein.