High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems

Abstract

Soybean agglutinin (SBA) is a specific N-acetylgalactosamine-binding plant lectin that can agglutinate a wide variety of cells. SBA has great potential for medical and biotechnology-focused applications, including screening and treatment of breast cancer, isolation of fetal cells from maternal blood for genetic screening, the possibility as a carrier system for oral drug delivery, and utilization as an affinity tag for high-quality purification of tagged proteins. The success of these applications, to a large degree, critically depends on the development of a highly efficient expression system for a source of recombinant SBA (rSBA). Here, we demonstrate the utility of transient and stable expression systems in Nicotiana benthamiana and potato, respectively, for the production of rSBA, with the transgenic protein accumulated to 4% of total soluble protein (TSP) in Nicotiana benthamiana leaves and 0.3% of TSP in potato tubers. Furthermore, we show that both plant-derived rSBAs retain their ability to induce the agglutination of red blood cells, are similarly glycosylated when compared with native SBA, retained their binding specificity for N-acetylgalactosamine, and were highly resistant to degradation in simulated gastric and intestinal fluids. Affinity column purification using N-acetylgalactosamine as a specific ligand resulted in high recovery and purity of rSBA. This work is the first step toward use of rSBA for various new applications, including the development of rSBA as a novel affinity tag for simplified purification of tagged proteins and as a new carrier molecule for delivery of oral drugs.

Fig. 1

Western blot analysis of transient protein expression of rSBA in Nicotiana benthamiana.a Western blot showing the accumulation of NbrSBA with (+) and without (−) p19, which was used in all subsequent transient assays. b Accumulation of rSBA from 1 to 9 days after infection (DAI). + is native SBA. c Accumulation of rSBA at day 5 with different A₆₀₀ concentrations of Agrobacterium. + is native SBA, NB is Nicotiana benthamiana wild-type TSP. d ELISA data for accumulation at days 1–9 after infection at optimized concentration. Values are expressed as a percentage of total soluble protein (TSP), with each bar representing the mean value of the three collected samples repeated in triplicate from each day, with standard error. All western blots were loaded with approximately 30 μg TSP loaded per lane, 150 ng native SBA control

Fig. 2

Purification of rSBA.a western blot showing the purification of NbrSBA using either His purification (H) or N-acetylgalactosamine (S). + is native SBA, TSP is total soluble protein prepared from Nicotiana benthamiana; FT, flow through; W, wash; Pur, purified. No signals were detected in FT-S. b Coomassie stained SDS–PAGE gel. Note the single band in Pur-S at 32 kDa, equivalent to the expected monomer size. Numbers on the left of the blots represent the sizes (kDa) of the protein ladder

Fig. 3

Hemagglutination/sugar inhibition study. Rabbit red blood cells undergo agglutination when treated with SBA, because of the presence of N-acetylgalactosamine on membrane-bound proteins. a Agglutination of rabbit red blood cells (RBC) following treatment with native SBA control or Nicotiana benthamiana-derived rSBA (NbrSBA), resulting in clumping around the bottom of the well. UT, untreated RBC, as characterized by RBC settling to the bottom of the well. bN-acetylgalactosamine inhibition. Both native SBA and Nicotiana benthamiana-derived rSBA are inhibited by N-acetylgalactosamine, enabling RBCs to pellet

Fig. 4

Determination of glycosylation of plant-derived rSBA. a Deglycosylation of NbrSBA and StrSBA using PNGase F, which cleaves N-linked glycans. Native SBA (+), NbrSBA, and StrSBA were treated by PNGase F, and analyzed by SDS–PAGE and western blotting. Untreated samples were used as controls. + is native SBA. b Concanavalin A binding analysis. Plant-derived rSBA and native SBA, both untreated and treated with PNGase F, were treated with ConA, which binds to N-linked mannose. ConA-treated samples were analyzed by SDS–PAGE and western blotting using mouse anti-ConA antibody. Binding to both untreated native and recombinant SBA, and no binding to PNGase F treated samples, suggests the presence of high mannose-type glycans for both recombinant and native SBA

Fig. 5

In-vitro digestion of Nicotiana benthamiana-derived rSBA in SGF and SIF.a NtrSBA is not degraded in simulated gastric fluid (SGF), even after incubation for 30 min (m), demonstrating resistance to both low pH and pepsin digestion. b NtrSBA is stable during simulated intestinal fluid (SIF) digestion. c and dE. coli-made hGAD65 is rapidly degraded in SGF and SIF, respectively. Each lane contains the starting equivalent of ~500 ng purified protein