His-tag fusion shown previously, degradation of Ub and especially SUMO fusions was alleviated somewhat in the Lon protease deficient E. coli strain BL21 pLysS, compared with that in native strain TG1. Quite unexpectedly, when ThiS-fused mRI was PP 242 custom synthesis induced in E. coli TG1 strain, the expressed fusion product was indiscernible at range of 50 to 66 kD in SDS-PAGE. An overexpressed band was noticed at around 25 kD in the inclusion bodies. Except the intense bands of smaller fragments as degradated products, only trace amount of product at the expected molecular weight was shown in Western blot. Since Lon was involved in mRI degradation 
for His, Ub and SUMO fusions, we explored the role of Lon, as well as HslV, another ATP-dependent protease in E. coli, in the breakdown of ThiS fusion of mRI. In all the protease-deficient hosts, degradation was not blocked or alleviated, as observed on immunoblot. Questions may be raised respecting the specificity of immunoblots, hence the possibility arises that immuno-reactive bands came from non-specific proteins rather than the degradated target protein. It seems unlikely since all the blots over this study showed clear background for cells without chemical induction, except that a small amount of leaky expression was exclusively observed for some target fusions. The overexpressed band of ThiS-mRI at around 25 kD was subjected to in-gel trysinization and MS analysis. It was identified as an N-terminal fragment of ThiS-mRI, thus verified as the degradated target protein instead of non-specific proteins. Ubl from both eukaryotes 7623957 and prokaryotes share similar tertiary structure with different primary structure. It was possible that a specific sequence or motif in ThiS, which is not present in other Ubl, was responsible for ThiS-directed breakdown of fusion target. We further explored which part of ThiS protein was 9504387 involved in the target degradation. The result in Fig. 3D indicated that both the N-terminal and C-terminal half-proteins conferred much less degradation than full length ThiS. It suggested that the whole ThiS Fusion for Heterologous Expression in E. coli 4 ThiS Fusion for Heterologous Expression in E. coli structure of ThiS rather than a single fragment was responsible for the protein degradation. 4. ThiS Fusion Enhanced the Expression of EGFP We further explored the effect of fusion on Green Fluorescent Protein expression. GFP is a highly stable protein that can be easily expressed in E. coli. We fused the gene encoding EGFP in frame but at upstream to the gene of ThiS and cloned into prokaryotic expression vector pQE30. This EGFP in fusion with ThiS at the Cterminus, was expressed in E. coli TG1 in inclusion bodies at 37uC, the same as EGFP protein alone without fusion. SDSPAGE of cell lysates indicated that the ThiS fusion product was expressed more abundantly and induced at earlier time than EGFP alone. Anti-His-tag immunoblot of the proteins revealed the overexpressed bands as the target proteins. Series of fast migrating smaller fragments were seen for both proteins in immunoblot but not in gel staining, that indicated a mild degradation of expressed products, which was more prominent for ThiS fusion than EGFP alone. Since both the cells expressing EGFP with and without ThiS fusion were fluorescent, suggesting that even the folded active proteins aggregated in inclusion bodies, we wondered if the enhanced expression of ThiS fusion was correlated with its improved foldability of EGFP in vivo. T
