Functional
Genome Analysis (B070)
Deutsches
Krebsforschungszentrum,
Im Neuenheimer Feld 580
D-69120
Heidelberg,
Germany. |
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Archive |
Proteomics
- Protein Microarrays |
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Combinatorial
peptide synthesis with laser-based
transfer of monomers
Laser writing is used to
structure surfaces in many different ways in materials and life
sciences.
However, combinatorial patterning applications are still limited. In a
collaborative project coordinated by colleagues at KIT, a method was
developed for cost-efficient combinatorial synthesis of
very-high-density peptide
arrays with natural and synthetic monomers. A laser automatically
transfers
nanometre-thin solid material spots from different donor slides to an
acceptor.
Each donor bears a thin polymer film, embedding one type of monomer.
Coupling
occurs in a separate heating step, where the matrix becomes viscous and
building blocks diffuse and couple to the acceptor surface.
Furthermore, two
material layers of activation reagents and amino acids can be deposited
consecutively.
Subsequent heat-induced mixing facilitates an in situ
activation and
coupling of the monomers. This allows to incorporate building blocks
with click
chemistry compatibility or a large variety of commercially available
non-activated, for example, post-translationally modified
building blocks into the array’s peptides with >17,000 spots per
square
centimetre.
Loeffler et
al. (2016) Nature Comm. 7,
11844. 
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  Production of
high-density protein-microarrays by cell-free in
situ expression
 
Due to the
success of DNA-microarrays and the growing numbers of available protein
expression clones, protein microarrays become more and more popular for
the
high-throughput screening of protein interactions. However, the
widespread
applicability of protein microarrays for this and other applications is
hampered by the large
effort
associated with their production. Beside the requirement for a protein
expression library, the actual protein expression and purification
represents bottleneck.
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As part of
the EU-funded MolTools
project (and several others thereafter), we
established
a process that allows the generation of protein microarrays from
process by which proteins are expressed from unbound DNA
template molecules on the microarray surface (or on any solid support).
It comprises the spotting of DNA
templates onto the surface and the transfer of a cell-free
transcription and
translation
mix on top of the same spot in a second spotting run. We demonstrated
the time and template dependence of this
coupled
transcription and translation and showed that enough protein is
produced to
yield signals that are comparable to 300 µg/ml of spotted
protein. Plasmids as
well as unpurified PCR-products can be used as templates and as little
as 35 fg
of PCR-product (~22,500 molecules) are sufficient for the expression of
full-length proteins.
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We
adapted the
system to the high-throughput expression of libraries by designing a
single
primer pair harbouring promoter, ribosomal binding
site and terminator sequences for an on
the chip expression of a multitude of such PCR-products. Utilising
full-length cDNA libraries of overall 16,000 human clones and
PCR-primers directed at all genes of various other organisms, we
are
producing such microarrays for various types of analysis.
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Utilising
the capability of detecting the interaction of individual
molecules and therefore being able
to count the actual number of interacting molecules, we are able to
perform
interaction studies in a really quantitative manner.
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Schmidt
et al. (2011) J. Prot. Res. 10, 1316-1322. |
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Angenendt
et al. (2006) Mol.
Cell. Prot. 5,
1658-1666. |
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Sobek et al. (2006) Comb.
Chem.
High-Throughput Screening 9,
365-380. |
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Kersten et
al. (2005) Expert
Rev. Proteomics 2, 499-510. |
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Angenendt, P. (2005) Drug
Discovery Today 10, 503-511. |
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