The sequence of a second member of the lima bean lectin gene family and the expression and characterization of recombinant lectin in Escherichia coli.

E.T Jordan,I.J Goldstein

doi:10.1016/s0021-9258(17)37340-4

E.T Jordan, I.J Goldstein

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https://doi.org/10.1016/s0021-9258(17)37340-4

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Abstract

The lima bean lectin recognizes terminal alpha-D-GalNAc groups and agglutinates human type A erythrocytes. We have cloned a portion of the gene encoding the alpha subunit of the lima bean lectin. The clone was obtained using the polymerase chain reaction and verified from a genomic clone encoding the mature protein of 253 amino acids. The deduced amino acid sequence has significant overall homology with other leguminous plant lectins and contains all of the known peptide sequences isolated 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 (rLBL) were obtained from Escherichia coli, using the T7 RNA polymerase expression system. SDS-polyacrylamide gel electrophoresis and Western blot analysis indicate expression of one protein band of about 27 kDa in induced E. coli cells. This protein cross-reacts with polyclonal antibodies raised against seed lectin (sLBL) and gave a reaction of identity with seed lectin by Ouchterlony double diffusion, specifically agglutinates type A blood cells, and 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', respectively). rLBL binds to hydrophobic ligands independent of sugar binding, an observation similar to results obtained with sLBL. However, despite the similar activities described, several significant differences between recombinant and native lima bean lectin were found, including 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.

Highlights

Milligram quantities of soluble active recombinant lima bean lectin(rLBL)were obtained from Escherichia coli, using the T7 RNA polymerase expression system
Cloning ofa GeneEncoding the Lima Berm Lectin-PCR was used to amplify the mature coding regionof lima bean lectin gene(&by using primers based upon previously reported amino acid sequences (3,27,28) as well as cDNA sequence (15)
The data presentedin this paper indicate the presence of a lectin gene family in I! lunatus and that we have cloned one gene from this family

Summary

Introduction

Milligram quantities of soluble active recombinant lima bean lectin(rLBL)were obtained from Escherichia coli, using the T7 RNA polymerase expression system. The crystallographic x-raystructures of several legume lectinhs ave been solved and have afforded crucial information regarding conserved structures(based upon conserved primary strucblot analysis indicateexpression of one protein bandof tures) of legume lectins and the molecular basis of lectin-carabout 27 kDa in induced E. coli cells Despite the similar activities described, several significant differences between recombinant and native lima bean lectin were found, including mobility on gel filtration, aggregation in solution, and itsCD spectrum. These differences may be due to a numbeorf factors, which will be discussed. The recombinant lectins, with few exceptions, have not been characterized sufficiently to distinguishpotential differences between the recombinant and native lectins

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