engine offers different types of Protein Macroarrays. The corresponding cDNA expression libraries were validated and advanced based on more than fifteen years of experience in the field.
hEX1 engine Protein Macroarray
The hEX1 protein expression library was constructed by preparing cDNA from human fetal brain poly(A)+ RNA, using oligo (dT)-priming. The resulting fragments (larger than 500 bp) were cloned into a modified expression vector (pQE30NST) allowing protein expression to be induced by addition of IPTG to the growth medium. The resulting expressed proteins include an N-terminal RGS-HIS-Tag, which can be used for detection and purification purposes. 193,536 clones were picked, spotted and induced on PVDF-membranes and thereafter assayed for protein expression using an anti-HIS-antibody. Based on this screen, 37,830 putative expression clones were selected and re-arrayed into 384 well microtiter plates which constitute the hEX1-library. Spotted on high-density protein filters on two PVDF-membranes, the library is available as hEX1 part 1 and part 2 (Büssow et al., NAR1998). Most clones of this library have been 5’tag sequenced and annotated. The clone collection contains full-length as well as shorter cDNA clones representing full-length along with partial proteins. Some clones express artificial sequences derived from cDNA-construct as well as 5’ and 3’ UTRs.
hEXselect engine Protein Macroarray
The hEXselect protein expression library was derived from the hEX1 library by in silico analysis and re-arraying. Genes which were available in a high clone number were reduced to decrease redundancy. Expressed proteins, which were available only as a single copy were doubled for successive production of high density protein macroarrays. The hEXselect protein expression library contains 23,806 clones. Like the hEX1-library, it comprises full-length as well as shorter cDNA clones. All clones have been 5’ tag sequenced and are fully annotated. The size of the low redundancy collection allows a complete spotting in duplicates onto a single Protein Array, which saves processing time and hybridisation probe volume. The majority of the clones represent partial proteins, which may include artificial protein sequences corresponding to the 5’UTR sequences, with one third matching to the human proteome. Protein Arrays are delivered with an accompanying annotation table and respective spotting positions.
UniPEx engine Protein Macroarray
The UniPEx protein expression library consists of 2 Arrays representing clones in 2 different modified vectors. In total, more than 100,000 sequenced clones from different protein expression libraries (human fetal brain, T-cells, lung) were analysed in depth for their coding potential. After in-frame analysis only clones with a confirmed in-frame ORF were selected and redundancy with respect to clones per gene was minimized to less than 3 fold. In total, 15,456 UniPEx clones represent 7,390 distinct human proteins.
Custom-made engine Protein Arrays
can be generated from subsets of these libraries in the range of 500 to 13,000 clones spotted in duplicates. The customer provides us with a list of desired clones/genes/fragments or even his own clones. Clones of the customer may have to undergo sub cloning, in order to be able to express protein on the array. We will then analyse the set and, if wanted, may offer some more/different clones to be placed on the array. We also can provide suitable controls (e.g. house keeping genes) to be included on the array. In agreement with the customer, we can then set up the custom-made Protein Array of the desired size.
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