SUMO fusion facilitates expression and purification of garlic leaf lectin but modifies some of its properties.
Over expression of lectin genes in E. coli often gives inclusion bodies that are solubilised to characterize lectins. We made N-terminal fusion of the Allium sativum leaf agglutinin (ASAL) with SUMO (small ubiquitin related modifier) peptide. The SUMO peptide allowed expression of the recombinant lectin in E. coli, predominantly in soluble form. The soluble fusion protein could be purified by immobilized metal affinity column (IMAC), followed by size exclusion chromatography. The SUMO protease failed to cleave the SUMO peptide from ASAL. This may be due to steric hindrance caused by the homodimer structure of the chimeric ASAL. Some properties like dimerization, haemagglutination and insecticidal properties of the recombinant SUMO-ASAL fusion protein were comparable to the plant derived native lectin. However, glycan array analysis revealed that the carbohydrate binding specificity of the recombinant SUMO-ASAL was altered. Further, the fusion protein was not toxic to E. coli (native ASAL exhibited toxicity). The recombinant lectin was more thermo-labile as compared to the native lectin. Three important findings of this study are: (1) sugar specificity of ASAL can be altered by amino-terminal fusion; (2) anti-E. coli activity of ASAL can be eliminated by N-terminal SUMO fusion and (3) SUMO-ASAL may be a preferred candidate insecticidal protein for the development of transgenic plants.
Upadhyay
Santosh Kumar
SK
National Botanical Research Institute, Council of Scientific and Industrial Research, Rana Pratap Marg, Lucknow 226001, India.
Saurabh
Sharad
S
Rai
Preeti
P
Singh
Rahul
R
Chandrashekar
K
K
Verma
Praveen C
PC
Singh
P K
PK
Tuli
Rakesh
R
D000371:Agglutination; D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism; D018744:DNA, Plant / Q000302:isolation & purification; D052978:Disk Diffusion Antimicrobial Tests; D005737:Garlic / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000187:drug effects; D037241:Mannose-Binding Lectins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D046228:Microarray Analysis; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D025842:SUMO-1 Protein / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera / Q000187:drug effects; D013696:Temperature
D018744:DNA, Plant; D037241:Mannose-Binding Lectins; D010940:Plant Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D003546:Cysteine Endopeptidases