Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1).
CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.
Varejão
Nathalia
N
Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Universidade Federal do Rio de Janeiro, Av. Bauhínia, 400, 21941-590, Rio de Janeiro, RJ, Brazil.
Almeida
Marcius da Silva
Mda S
De Cicco
Nuccia N T
NN
Atella
Geórgia C
GC
Coelho
Luana C B B
LC
Correia
M A Tereza S
MA
Foguel
Debora
D
D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007887:Fabaceae / Q000737:chemistry*; D006386:Hemagglutination Tests; D037121:Plant Lectins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014349:Trypanosoma cruzi / Q000378:metabolism
D037121:Plant Lectins; D011994:Recombinant Proteins