The currently available Mammalian Gene Collection clones available through ATCC serve as the pool of candidate cDNAs for human protein production. Rather than attempt to express every human cDNA, as will be done with P. furiosus and C. elegans, the basic experimental protocol for human protein production will include an initial screen of cDNA sequences to catagorize proteins. This computer based process will be used to screen for 1) molecular weight, 2) pI, 3) the possession of organelle targeting motif sequences, 4) the possession of putative transmembrane segments, 5) presence of possible sites of glycosylation, and 6) possession of identifiable cofactor binding motifs. Sequences will also be subjected to "threader" analysis to determine if strong similarities exist with any structurally characterized protein. Previously crystallized proteins will be eliminated and efforts will be made to include as many classes of unknown proteins and enzymes as possible in the subsequent expression screening process.

Following screening, the pool of candidate cDNAs for initial high-throughput expression and purification will be reduced by the temporary elimination of putative transmembrane proteins. Remaining cDNAs will be cloned into a custom TOPO (Invitrogen) vector. This vector contains an amino terminal six his tag under the control of the Trc (Trp + Lac) promoter, with epitopes removed so as to minimize the introduction of large vector-encoded tags. The initial choice of this vector was based upon our experience in the successful expression and production of thirty soluble and membrane associated proteins, many of which contained [2Fe-2S] clusters or flavin cofactors.
To express proteins, one liter cultures of E. coli containing the desired cDNA in the expression vector are grown in Circlegrow (Qbiogene) medium at 30 deg. C and harvested after entering stationary phase, at which time the Trc promoter is maximally induced. A key feature of this expression protocol is that no culture monitoring or IPTG induction is required. Cells are harvested, resuspended in buffer containing 0.2% sodium cholate (to increase the solubility of membrane associated and poorly soluble proteins), and sonicated . After ultracentrifugation to remove cell debris, the supernatant is passed through a TALON (Clontech) metal chelate column, washed with column equilibration buffer, and eluted with buffer containing 300mM imidazole. The eluted protein fraction is immediately passed onto a size exclusion column. Protein expression and purity is determined by SDS PAGE and UV-Vis spectra.

For long term interests, it is desirable to consider production of "difficult" proteins and to develop strategies that will eventually allow production of all proteins. Towards this end we have actively examined new, more effective expression vectors for production of human proteins. We found those vectors currently available for bacterial expression to be less, or no more, effective for difficult proteins than our current Tac-based expression vector. However, we have recently produced a new expression vector that is based upon the Tac promoter but is a fusion with a newly-identified human protein that has given significantly increased yields for a number of test proteins. Inclusion of a TEV protease cleavage site also allows production of a protein without a tag. This system will become our first backup for difficult to express proteins.

We also propose to conduct secondary screening of proteins that do not express well in procaryotic systems. cDNAs that resulted in poor expression in initial attempts will be moved into different expression vectors, or into eucaryotic expression systems such as COS and MEL (murine erythroleukemia) cells. This system has great potential to produce properly processed mammalian proteins in significant yield if we can successfully design a convenient vector system for cassette insertion of cDNAs of interest. We will also plan to investigate in vitro translation as a potential source of target proteins.

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