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Following the completion of genome sequencing projects,
protein chips are emerging with the potential to add functional
flesh to the bare bones of encoding genes and thus leading the way
to rapidly profile the entire proteome. However, compared with DNA
chips, protein chips provide more challenge due to the complexity
and inherent difficulties. Such as, like DNA, it is not possible to
amplify proteins and thus to detect very tiny amounts; it is not
easy to attach the proteins to chips; it is not easy to maintain
their integrity and stability as proteins tend to adsorb
non-specifically to surfaces and leading the possibility of
denaturation and loss-of-function. In addition, the display of
proteins in microarray format is a problem for which there is no
general solution yet. Therefore, to foster these challenges, we are
focusing on engineering and converging microarray surfaces and
RNA-protein fusion techniques to create high-throughput functional
protein arrays directly from encoding mRNA, such that the encoded
proteins are immobilized on a surface as they are synthesized.
Solid-phase cell-free Protein factory:
From protein synthesis to Proteomics
Cell-free systems have proved to have high utility at the
genomic, transcriptomic and proteomic levels but despite the
encouraging results from advanced cell-free translation systems,
there are less efforts on improving the yields of stable and
functionally active proteins and what is crucial for modern
proteomic microarray methods. In this research, we are developing a
joint and novel solid-phase approach to cell-free translation and
mRNA-protein fusion techniques that could simultaneously synthesize,
immobilize and stabilize proteins onto solid surfaces using anchored
mRNA. We discovered that the proteins synthesized in such-a-manner
adopt a more native state and thus the more biological activity in
comparison with the conventional liquid-phase approaches (Nucleic
Acids Research, 2006). In
contrast to the cell-free liquid-phase system, ribosomes are bound
to the endoplasmic reticulum inside the living cells that would
promote protein maturation and translocation. The solid-phase
approach shown herein however, controls cell-free protein synthesis
reaction in a similar stationary mode using anchored mRNA, and thus
it would help to direct protein folding which is one of the most
important processes in biology. Moreover, it also improves the half
life of the mRNA molecule, which is very short in cell-free systems,
by protecting its 3’-terminus against contaminating nucleases.
HTP Microarray Evolution Reactor: a novel
concept of “single gene-encoded DNA-to-Protein chip”
In
this research, a novel microarray concept of ‘single molecule-type
RNA-to-protein chip’ is introduced in which each nano-well
represents a single molecule-type mRNA-protein complex, where mRNA
provide genetic information about the functionally active protein
which is insured to retain their correctly folded functional
content. A realistic scheme is designed by combining the potential
of a group of approaches: single-molecule dilution (SMD),
solid-phase RNA-protein fusion, and microarray. To build the
platform, we have fabricated microreactor array chips comprising of
uniformly distributed sub-picoliter scale reactors of 6µm in
diameter. As a model experiment, in this research we are attempting
to screen a library generated by replacing S65 to random cDNA
sequences of wtGFP.
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