Cloning and soluble expression of mature α-luffin from Luffa cylindrica in E. coli using SUMO fusion protein

Abstract

α-Lufin, found in Luaf cylindrica seeds, is a type I ribosome inactivating proteins. Cytotoxic effects make it an appropriate candidate for the construction of immunotoxins and conjugates. Because of limited natural resources, recombinant technology is the best approach to achieve large-scale production of plant-based proteins. In the present study, α-lufin protein was expressed in E. coli and the effects of different temperature conditions, SUMO fusion tag, and cultivation strategies on total expression and solubility were investigated. Protein expression was evaluated at different intervals (0, 4, 6, 8, 24 h) postinduction. Our results showed that EnBase had higher eficiency than LB, and maximum solubility and total protein expression were achieved 24 h after induction at 30 °C and 25 °C, respectively. It was shown that SUMO tag is an effective strategy to improve protein solubility.

Keywords: E; coli expression system; fed-batch culture; ribosome inactivating proteins; soluble expression; α-lufin.

Figure 1

Analysis of pFUSE pRham N-His SUMO-α-luffin recombinant plasmid by restriction enzyme digestion. A) According to the plasmid map, predicted product sizes were 1364 and 1934 bp. B) Gel-electrophoresis analysis of the pFUSE pRham N-His SUMO-α-luffin plasmid by EcoRV restriction enzyme digestion on a 0.8% agarose gel. Lane 1: digested N-His SUMO-α-luffin; lane 2: undigested N-His SUMO-α-luffin; lane 3: 1 kb DNA markers.

Figure 2

PCR analysis of extracted recombinant plasmids from kanamycin-resistant E. coli cells. PCR products were electrophoresed on a 0.8% agarose gel. Lanes 1 and 2: amplification of α-luffin gene; lane 3: negative control; lane 4: 1 kb DNA markers.

Figure 3

SDS-PAGE analysis of α-luffin expression in LB medium. Lane 1: Cell lysates before rhamnose induction; Lane 2: Cell lysates 4 h postinduction; Lane 3: Cell lysates 6 h postinduction; Lane 4: Cell lysates 8 h postinduction; Lane 5: Cell lysates 24 h postinduction; Lane 6: Fermentas Protein marker SM0671.

Figure 4

Western blot analysis of recombinant His tag-α-luffin. Lane 1: 42 kDa His tagged α-luffin; Lane 2: His tagged protein as positive control; Lane 3: Fermentas protein marker SM0671.

Figure 5

SDS-PAGE analysis of expressed recombinant SUMO/α-luffin at different temperatures and times in EnBase medium. The samples were separated on 12% resolving gel under reduced condition. The gels were stained with Coomassie Blue. A) Coomassie stained gel under reduced condition, EnBase medium at 37 °C. Lane 1: Cell lysates before rhamnose induction; Lane 2: Cell lysates 4 h postrhamnose induction; Lane 3: Cell lysates 6 h postrhamnose induction; Lane 4: Cell lysates 8 h postrhamnose induction; Lane 5: Cell lysates 12 h postrhamnose induction; Lane 6: Cell lysates 24 h postrhamnose induction; M: Fermentas protein marker SM0671. B) Coomassie stained gel under reduced condition, EnBase medium at 30 °C. Lane 1: Cell lysates before rhamnose induction; Lane 2: Cell lysates 4 h postrhamnose induction; Lane 3: Cell lysates 6 h postrhamnose induction; Lane 4: Cell lysates 8 h postrhamnose induction; Lane 5: Cell lysates 12 h postrhamnose induction; Lane 6: Cell lysates 24 h postrhamnose induction; M: Fermentas protein marker SM0671. C) Coomassie stained gel under reduced condition, EnBase medium at 25 °C. Lane 1: Cell lysates before rhamnose induction; Lane 2: Cell lysates 4 h postrhamnose induction; Lane 3: Cell lysates 6 h postrhamnose induction; Lane 4: Cell lysates 8 h postrhamnose induction; Lane 5: Cell lysates 12 h postrhamnose induction; Lane 6: Cell lysates 24 h postrhamnose induction; M: Fermentas protein marker SM0671.

Figure 6

SDS-PAGE analysis of soluble and insoluble expression of recombinant SUMO/α-luffin expression at different temperatures and times in EnBase medium. The samples were separated on 12% resolving gel under reduced condition. The gels were stained with Coomassie Blue. The distribution of total cell lysates, pellet and soluble fractions during different time intervals post rhamnose induction. B = before induction by rhamnose, T = total lysate, P = pellet, S = supernatant or soluble fraction, M: Fermentas protein marker. A) Coomassie stained gel under reduced condition. The distribution of total cell lysates, pellet and soluble fractions during different time intervals post rhamnose induction at 37 °C in EnBase medium. B) Coomassie stained gel under reduced condition. The distribution of total cell lysates, pellet and soluble fractions during different time intervals post rhamnose induction at 30 °C in EnBase medium. C) Coomassie stained gel under reduced condition. The distribution of total cell lysates, pellet and soluble fractions during different time intervals post rhamnose induction at 25 °C in EnBase medium.