Fig. 5

Verification of processing of human proteins by HERV-K(HML-2) Protease in vitro. Human candidate proteins were expressed in vitro using a coupled transcription/translation system. a. Results from protease incubations of various candidate proteins labeled with either ³⁵S-methionine or a C-terminal HA-tag (“³⁵S” and “HA”) are shown. Experiments included for each candidate protein a reaction without protease (“C”), one with protease (“+”), and one with protease and Pepstatin A (“+/P”). Reaction products were separated by SDS-PAGE in 10% PAA-gels and processed for phosphorimager analysis or HA-tag-specific Western blots depending on the label. Processing of full-length candidate proteins (indicated by an arrow) was evidenced by additional protein bands smaller than the respective full-length candidate protein (arrowheads) and/or a decrease in the amount of full-length candidate protein (see the Results section). One example of a candidate protein (PSMC4) without evidence of processing by HML-2 Pro is shown. b. Graphical depiction of candidate proteins confirmed to be processed by HML-2 Pro. The number of amino acids and corresponding molecular mass in kDa is indicated by scales at the top and by the line length for each protein. Positions of methionines and cleavage sites (grey and black arrowheads, respectively), as identified by TAILS in either one of the two replicate experiments at pH 5.5 (see the text), are indicated for each protein. Dashed lines indicate molecular masses of processing products observed experimentally for either ³⁵S-methionine (“³⁵S”)- or HA-tag (“HA”)-labeled candidate proteins. Note that the latter label will only detect C-terminal processing products. Processing products were not indicated for the two HSP90A proteins because observed products were difficult to assign due to too many observed cleavage sites. Processing of PDIA3 protein was supported by reduction of the amount of full-length protein, though no smaller processing products could be observed. Note that C15orf57 migrated slower in gel electrophoresis than predicted by molecular mass. See Additional file 2: Figure S2 for additional evidence of processing of candidate proteins by HML-2 Pro