Isolation, characterization and production of a new recombinant lectin protein from leguminous plants

Abstract

Lectins are present in microorganisms, plants and animals and have attracted great interest due to their varied physiological roles in cell agglutination, anti-tumour, immunomodulatory, antifungal and antiviral effects. Legume lectins are important to the pharmaceuticals but they are produced in low amounts in the plant seeds. Moreover, the genes controlling these proteins are conditionally active, i.e., they work under specific circumstances and not in regular manner. Looking into these limitations, we aimed this work to produce a recombinant lectin for pharmaceutical use especially for the treatment of cancer. Three different legume plant species were collected from Aseer region of Kingdom of Saudi Arabia viz., Acacia seyal, Pisum sativum (wild type) and Pisum sativum (Pea). The plant tissues were subjected to RNA extraction, the extracted RNA was used for lectin gene amplification using specific primers. Cloning, subcloning of the Acacia 400bp gene was carried out and in vitro transcription, combined with protein purification was undertaken. The cytotoxicity of the recombinant lectin was performed on two cell lines such as breast cancer MCF-7 and liver cancer HepG-2 cells. Our study resulted in the observation of two amplicones with all the three examined species, the amplicone molecular sizes were 800 and 400bp. The 400bp amplicone was excised from the agarose gel, purified and sequenced. The sequence analysis revealed that the nucleotide sequence belongs to lectin gene. The sequence analysis revealed that the lectin gene isolated from Pisum sativum (wild plant) is similar to the Pisum sativum lect1 with identity 90%, whereas, lectin isolated from Pisum (pea) showed similarity of 91% with the other lectins. On the other hand, Acacia lectin showed similarity with Lotus japonicus nod factor binding lectin gene with identity of 95%. Thus we conclude that new lectin protein of 17 and 15 kDa was produced that can be used by pharmaceutical industries.

Pea, Pisum sativum, and its anticancer activity

“…The active phytochemical substances of P. sativum are as follows: Asparaginase;[ 9 ] flavonoids including apigenin, daidzein, genistein, and kaempferol;[ 10 ] lectin;[ 11 12 13 ] phenolic compounds including caffeic, catechin, coumaric acids, gentisic acids, ferulic, protocatechuic, and vanillic acids;[ 14 15 ] pisatin and an allelopathic active substances;[ 16 17 ] proanthocyanidin;[ 18 ] saponins;[ 19 20 ] steroid phytohormone including brassinosteroid;[ 21 22 ] and tannins. [ 23 ]…”

“…[ 43 ] studied the P. sativum extracted lectins from Egypt exhibited the antiproliferative property to liver cancer cell line. Patel[ 12 ] extracted lectin from leaves and buds of P. sativum from Saudi Arabia and studied cytotoxicity to many cancer cell lines such as MCF-7 (breast), HepG-2 (liver), HEP-2 (larynx), and HCT-116 (colon). In recently, Stanisavljevic et al .…”

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“…Lectin and maltose caused significant in liver enzymes. However, phosphate enzymes act by hydrolyzing phosphomonoester including 3, and 5, phosphoproteins and these may also be involved in the transfer of phosphate, phosphatases are involved in many different processes that require mobilization of phosphate ion or dephosphorylating as part of anabolic, catabolic or transfer processes 5,10 .…”

“…Lectins are present in a diversity of organisms including microorganisms, flowering plants, animals, fungi, and bacteria 5 , and have attracted great interest due to their varied physiological roles in cell agglutination, antitumour, immunomodulatory, antiproliferative, antitumor, antifungal and antiviral effects 5,6 . They serve as powerful tools in immunological studies and can be employed as immunohistochemistry markers in diagnosis of cancer and profiling of cell surface types due to expression of aberrant glycans on diseased and transformed cells 4 .…”

Expression of Lectins in Heterologous Systems

“…Obtaining heterologous proteins could facilitate the cost-effective production of bioactive lectins for pharmaceutical purposes [ 56 ]. These systems offer higher yields than conventional purification methods, reducing costs and time.…”