*4.2. Construction of Recombinant Plasmids, Protein Expression and Purification*

Expression plasmid encoding CGL mutants was constructed as described earlier [10] on the basis of pET40/CmAP plasmid which carried the gene of alkaline phosphatase CmAP as a reporter gene. CGL mutants were genetically engineered by oligonucleotide-specific mutagenesis approach. The amino acid substitutions were introduced into the forward and reverse gene-specific primers (Table 2).


**Table 2.** Primers for construction of the recombinant plasmids.

The resultant mutant genes were amplified with the primers CGL-dir: 5 -AGCTGAGCTCGATG ACGATGACAAGATGACAACGTTTCTTATCAAACACAAGGCCAGTG-3 and CGL-rev: 5 -AGC TGTCGACTTAGGCATAAACTAAAACGCGCTTGTCTTT-3 , and ligated with the vector of pET-40b(+)/CmAP linearized by endonucleases SacI and SalI. The correct CGL cDNA sequence was verified by sequencing with ABI Prism Big Dye Terminator 3.1 Cycle Sequencing Kit and ABIPrism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Recombinant lectins were expressed in *E. coli* Rosetta (DE3) and purified as described previously [10].
