THE SEQUENCE OF A 2ND MEMBER OF THE LIMA-BEAN LECTIN GENE FAMILY AND THE EXPRESSION AND CHARACTERIZATION OF RECOMBINANT LECTIN IN ESCHERICHIA-COLI

The lima bean lectin recognizes terminal alpha-D GalNAc groups and agg lutinates human type A erythrocytes. We have cloned a portion of the g ene encoding the (alpha subunit of the lima bean lectin. The clone was obtained using the polymerase chain reaction and verified from a geno mic clone encoding the mature protein of 253 amino acids. The deduced amino acid sequence has significant overall homology with other legumi nous plant lectins and contains all of the known peptide sequences iso lated from lima bean lectin (LBL). Southern blot analysis reveals the presence of several genes which hybridize to the cloned gene and which we propose are genes included in the lima bean lectin gene family. We report here the sequence, expression, and characterization of LBL 2, the second member of this gene family. Milligram quantities of soluble active recombinant lima bean lectin (LBL) were obtained from Escheric hia coli, using the T7 RNA polymerase expression system. SDS polyacryl amide gel electrophoresis and Western blot analysis indicate expressio n of one protein band of about 27 kDa in induced E. coli cells. This p rotein crossreacts with polyclonal antibodies raised against seed lect in (sLBL) and gave a reaction of identity with seed lectin by Ouchterl ony double diffusion, specifically agglutinates type A blood cells, an d is specifically inhibited by D-GalNAc. The isoelectric point of rLBL is 5.86, whereas those of the seed lectin subunits were determined to be 5.86, 5.58, and 5.20 (previously designated alpha, beta, alpha(r), respectively). rLBL binds to hydrophobic ligands independent of sugar binding, an observation similar to results obtained with sLBL. Howeve r, despite the similar activities described, several significant diffe rences between recombinant and native lima bean lectin were found, inc luding mobility on gel filtration, aggregation in solution, and its CD spectrum. These differences may be due to a number of factors, which will be discussed.