Figure 1.

Cloning of the gene of Jacalin in single chain. (A) From total RNA of Jackfruit seed, cDNA synthesis was carried out with oligo-dT as primer, followed by PCR amplification of Jacalin gene by vent DNA polymerase using an upstream gene-specific primer containing NcoI site and a downstream gene specific primer containing an EcoRI site. The ATG sequence present in the NcoI site (ccATGg) acts as the initiation codon for the translation of the mature Jacalin polypeptide. Temperature conditions used for PCR amplification were 1 min at 95°C for denaturation, 1 min at 60°C for annealing, and 3 min 30 sec at 72°C for extension. At the end of 25 cycles, an additional 10 min at 72°C was given for extension. (B) The above amplified gene product had one internal NcoI site at 15th amino acid position, which was removed by silent mutation through site directed mutagenesis using internal Nco I knockout primer. Unlike the native protein, α and β chain of rJacalin are linked with T-S-S-N loop as indicated in the figure, to make a single chain protein. (C) The above mutated PCR product was cloned between NcoI and EcoRI site in the pT₇Nc vector described by Vandana et al. earlier (1997).