ProCA32.CXCR4 was constructed by engineering a CXCR4-targeting moiety (LGASWHRPDKFCLGYQKRPLP) to the C terminus of ProCA32; PEGylation was performed for surface modification. ProCA32.CXCR4 was expressed in BL21 (DE3) pLysS cell strain and purified following our established protocol (20). Two site-specific PEGylations, cysteine PEGylation and lysine PEGylation, were used for ProCA32.CXCR4 surface modification. For cysteine PEGylation, ProCA32.CXCR4 solution [concentration between 1 and 10 mg/ml, in 10 mM HEPES (pH 7.2)] was degassed by bubbling with nitrogen. Tris (2-carboxyethyl) phosphine hydrochloride (Sigma-Aldrich) solution was used to reduce disulfide bonds at room temperature for 20 min. Methoxy PEG maleimide (JenKem Technology) with a molecular weight of 2 kDa was reacted with reduced ProCA32.CXCR4 at a molar ratio of 1:1 overnight at 4°C. For lysine PEGylation, ProCA32/ProCA32.CXCR4 solution [concentration between 1 and 10 mg/ml, in 10 mM HEPES (pH 7.2)] was reacted with methoxy PEG succinimidyl carboxymethyl ester reagent (molecular weight of 2 kDa, JenKem Technology) at a molar ratio of 1:5 overnight at 4°C. Purification of the PEGylated protein sample was achieved by fast protein liquid chromatography. The PEGylation product was evaluated with Coomassie blue staining and iodine (I2) staining (fig. S1).

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