Immobilization Staphylococcal Protein A on Magnetic Cellulose Microspheres for IgG Affinity Purification

Figures & data

Figure 1 Construction of recombinant plasmid pET-SPA. The SPA gene was cloned under the downstream of T7 promoter in pET 21a (+) expression vector, which also contained the gene for ampicillin resistance.

Figure 2 (A) SDS-PAGE and (B) western blot analysis of purified SPA. Lane M: low range protein marker; Lane 1: control E. coli BL21 without recombinant plasmids; Lane 2 and 4: induced E. coli BL21 transformed with recombinant plasmid pET-SPA; Lane 3 and 5: affinity purified SPA.

Figure 3 ESEM micrographs of MCMS (× 1500).

Figure 4 Amount and recovery of IgG captured to the SPA-MCMS as a function of increased amounts of IgG added. The amount of rabbit IgG and serum in the sample was increased, as the total reaction – volume was kept at 1 mL. 100 µL of SPA-MCMS were used per sample. ▪and □ indicated amount of IgG captured from rabbit IgG and serum; ♦ and □ indicated recovery from rabbit IgG and serum.

Figure 5 Binding of IgG at different concentration and time. The total amount of IgG added was fixed to 1 mg. 100 µL SPA-MCMS were used per sample.

Table 1. Binding capacity of SPA-MCMS for various types of IgGs

IgG origin	Binding capacity (mg/mL)^a
Rabbit	10.83 ± 0.18
Mouse	9.26 ± 0.15
Rat	1.83 ± 0.02
Goat	3.48 ± 0.05
Human	9.12 ± 0.12

^aData represent the means ± SD of three independent experiments.

Table 2. Purification of rabbit IgG by SPA-MCMS from rabbit serum^a

	Protein^b (mg)	IgG^c (mg)	Purity (%)	Recovery (%)
Serum	135.10 ± 2.31	11.82 ± 0.21	8.75	–
Purified product	8.77 ± 0.16	8.65 ± 0.17	98.63	73.18

^aAll determinations were repeated three times by 1 mL of SPA-MCMS.

^bThe value of protein was determined by Lowry Method (means ± SD).

^cIgG was evaluated by sandwich ELISA (means ± SD).

Figure 6 HPSEC analysis of rabbit IgG purified by SPA-MCMS.