*2.7. Separation and Purification of Mal-mPEG5000-SPA*

Mal-mPEG5000-SPA was separated and purified by AKTA purifierTM10 with SuperdexTM75 gel column (Figure 7a–c). Separated by SuperdexTM75 gel column, there were three protein peaks: S1, S2 and S3 (Figure 7a), and the eluents in the collection tubes corresponding to the peak position were collected and determined for enzymatic specific activity. The specific activity of S1 and S2 was (1.245 ± 0.047) × <sup>10</sup><sup>4</sup> U/mg and (0.533 ± 0.036) × 104 U/mg respectively as shown in Figure 7c. As the peak of SPA elution was noted around 17 min according to the result of the pre-test, the elution peak S1 was collected, centrifuged and concentrated for further purification.

Mal-mPEG5000-SPA was separated for the second time using SuperdexTM75 gel column, and there were elution peaks S11 and S12 (Figure 7b). The specific activity of S11 and S12 was determined to be (1.422 ± 0.057) × <sup>10</sup><sup>4</sup> U/mg and (0.061 ± 0.009) × <sup>10</sup><sup>4</sup> U/mg respectively (Figure 7d). According to the separation principles of gel column chromatography, different positions of the peaks suggest different molecular weight of the protein under each peak. The position of SPA peak was around 17 min, while the position of Mal-mPEG5000-SPA was about 7 min, which indicated change in molecular weight as SPA was modified by Mal-mPEG5000.

**Figure 7.** Separation curve of Mal-mPEG5000-SPA by gel column SuperdexTM75 (**a**)Elution profile of Mal-mPEG5000-SPA by SuperdexTM75 for the first time; (**b**) Elution profile of Mal-mPEG5000-SPA by SuperdexTM75 for the second time; (**c**) The specific activity of the elution fractions from SuperdexTM75 for the first time; (**d**) The specific activity of the elution fractions from SuperdexTM75 for the second time.

SDS-PAGE gel electrophoresis was carried out on the collected elution peaks S1 and S11. The results were shown in Figure 8a,b. An obvious band of S1 was found above the SPA band as indicated in Figure 8a. The band was of Mal-mPEG5000-SPA, whose molecular weight was about 67 kDa compared with the standard marker. Figure 8b demonstrated the electrophoretogram of elution peak S11. A single band was noted after separation twice by SuperdexTM75, and Mal-mPEG5000-SPA was obtained through purification.

**Figure 8.** SDS-PAGE electrophoresis spectra of Mal-mPEG5000-β-SPA (**a**) separation by SuperdexTM75 for the first time; Lane 1: marker proteins; Lane 2: SPA; Lane 3: Mal-mPEG5000-SPA by SuperdexTM75. (**b**) separation by SuperdexTM75 for the second time; Lane 1: marker proteins; Lane 2: SPA; Lane 3: Mal-mPEG5000-SPA by SuperdexTM75.
