Annual Report 2011 - Center for Advanced Biotechnology and ...

Human transcription factors are typically complex, multidomain proteins, and in
terms of numbers of unique genes represent roughly 10% of the entire human
proteome. Our goal is to express native, folded, individual domains from these
different transcription factors with the hope that optimal specificity and affinity may
be obtained by raising affinity capture reagents agains 3D epitopes. In the first year
the Rutgers group has designed single-domain expression constructs using advanced
“domain parsing” software developed by Dr. Janet Huang and Prof. Gaetano T.
Montelione (see figure below). Together with Dr. Thomas Acton and Prof.
Montelione, we have also developed a new series of high-efficiency E. coli vectors
using “transcript optimized expression-enhancement technology” (“TOEET” – patent
applied for). These vectors enable unprecedented levels of expression and solubility
to be achieved. In 2011 the group cloned domains from nearly 1400 separate human
transcription factors in >2700 distinct constructs and expression-tested approximately
80% of these at small scale. We are now moving on to scale-up and production of
multi-milligram quantities of purified antigen for our affinity capture reagent partners.
In December, 2011, we shipped our first batch of purified antigens.
Fig. 1. Example of a portion of the output of the DisMeta server and how it can
be used to analyze transcription factor sequences for structural information.
Shown schematically is a portion of the sequence of a human transiption factor, IF –
16 (γ-interferon-inducible protein 16), analyzed by the DisMeta server.
In this segment of the sequence the server predicts two regions that
appear to have a complex, folded, tertiary structure separated by a
region of polypeptide chain that is predicted to be relatively disordered.
Three-dimentional structures for domains represented by the two
ordered regions shown were determined by the NESG. The leftmost
structure (PDB ID 2oq0) is a HIN-200/IF120x domain (InterPro ID
IPR004021), and the rightmost structure (PDB ID 3b6y) is a pyrin
domain (InterPro ID IPR004020) (Liao et al, unpulished). This example
illustrates how the DisMeta server can be used to scan TF protein
sequences to find the regions encoding domains that are likely to have
3D epitopes.
41