Fig. 1. The plasmid pET28a-m map. Underlined letters are primers, the forward primer contains a NcoI site and the extra ‘AC’, the reverse primer contains a XhoI site and the natural stop codon ‘TAA’.

Fig. 2. (a) Expression of rMuMIG in E. coli BL21. Lane M, Low Mw protein markers. Lane 1, uninduced E. coli cell lysate. Lane 2, E. coli lysate after IPTG induction. Lane 3, supernatant of E. coli lysate after ultrasonication of the induced E. coli. Lane 4, the pellet fraction of the ultrasonication of the induced E. coli. (b) Western blot analysis of E. coli lysate with commercial anti-MIG antibody. Lane 1, uninduced E. coli cell lysate. Lane 2, E. coli cell lysate after IPTG induction.

Fig. 3. (a) Purification profile of rMuMIG using S-Sepharose column. UV absorbance at 280 nm and conductivity were detected real-time. Two protein elution peaks were observed (Peak 1 and 2). (b) SDS–PAGE analysis of Peak 2 proteins. Lane M, Low Mw protein markers. Lane 1, the eluted rMuMIG protein at Peak 2.

Fig. 4. (a) Five micrograms of Peak 2 elute was loaded on SDS–PAGE, no other bands could be observed except the main band of rMuMIG. (b) Capillary electroporesis of rMuMIG. There was only one peak was observed at 9 min position and the peak value was 60 mAU. Running condition: bare fused-silica capillary with an effective length of 20 cm, 75 μm I.D; phosphate buffer of 40 mM, pH 11.0; separated at 10 kV; detected at UV214 nm.

Fig. 5. Chemotaxis of rMuMIG for mouse spleen lymphocytes. X-axis, rMuMIG concentrations in the bottom wells. Y-axis, percentage of total cells transmigrated through the transwell membrane. ^*p < 0.001 was calculated comparing to the negative control and ^#p < 0.002 was comparing to 100 ng/ml concentration by one-way ANOVA.

Fig. 6. rMuMIG inhibits bone marrow mononuclear cells transitioning into S-phage of cell cycle in vivo. DNA contents of bone marrow mononuclear cells were analyzed by FACS and percentages of cells in G₁, S, and G₂ phases of cell cycle were illustrated. Data from a representative mouse is shown in two histograms: rMuMIG-, the PBS control group, rMuMIG+, the mice received rMuMIG injection as described in Materials and methods. The DNA content (PI) is expressed in the FL-A channel.

Purification of recombinant mouse MIG using the E. coli expression system

Results are derived from 800 ml cultures of E. coli expressing the recombinant proteins. Total protein was estimated by the method of Bradford [13].