Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR products.
Due to the success of DNA microarrays and the growing numbers of available protein expression clones, protein microarrays have become more and more popular for the high throughput screening of protein interactions. However, the widespread applicability of protein microarrays is currently hampered by the large effort associated with their production. Apart from the requirement for a protein expression library, expression and purification of the proteins themselves and the lacking stability of many proteins remain the bottleneck. Here we present an approach that allows the generation of high density protein microarrays from unbound DNA template molecules on the chip. It is based on the multiple spotting technique and comprises the deposition of a DNA template in a first spotting step and the transfer of a cell-free transcription and translation mixture on top of the same spot in a second spotting step. Using wild-type green fluorescent protein as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein was produced to yield signals that were comparable to 300 microg/ml spotted protein. Plasmids as well as unpurified PCR products can be used as templates, and as little as 35 fg of PCR product ( approximately 22,500 molecules) were sufficient for the detectable expression of full-length wild-type green fluorescent protein in subnanoliter volumes. We showed that both aminopropyltrimethoxysilane and nickel chelate surfaces can be used for capture of the newly synthesized proteins. Surprisingly we observed that nickel chelate-coated slides were binding the newly synthesized proteins in an unspecific manner. Finally we adapted the system to the high throughput expression of libraries by designing a single primer pair for the introduction of the required T7 promoter and demonstrated the in situ expression using 384 randomly chosen clones.
Angenendt
Philipp
P
Functional Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany. p.angenendt@gmx.de
Kreutzberger
Jürgen
J
Glökler
Jörn
J
Hoheisel
Jörg D
JD
D002474:Cell-Free System; D020099:Coated Materials, Biocompatible; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009532:Nickel / Q000737:chemistry; D016133:Polymerase Chain Reaction; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D014176:Protein Biosynthesis; D012821:Silanes / Q000737:chemistry; D014158:Transcription, Genetic
D020099:Coated Materials, Biocompatible; D012821:Silanes; D049452:Green Fluorescent Proteins; D009532:Nickel; D004247:DNA; C071717:trimethoxysilane