Studies Of Recombinant Preparation And Inhibitor Screening Of Two Biological Target Proteins

Recombinant preparation and inhibitor screening of biological target proteins have a very important position for the prevention of many diseases. In this article, polygalacturonase from Apolygus lucorum and Glutamic-oxaloacetic transaminase from cytoplasm of human cells were chosen as research objects, and then, recombinant preparation and inhibitor screening of these two enzymes were explored. Polygalacturonase is a key enzyme for collapsing plant cell wall secreted by saliva of Apolygus lucorum, and therefore, this enzyme was thought as one of the plant pathogenic factors. Inhibiting the activity of polygalacturonase may reduce the damaging effects of insects. Aspartate aminotransferase from cytoplasm of human cells is a key enzyme in energy supplying of cancer cells, inhibiting the activity of aspartate aminotransferase may suppress the growth of cancer cells.1. Cloning and Bioinformatics Analysis of polygalacturonase gene of Apolygus lucorumA polygalacturonase gene AL-PG was cloned from the adults of the Apolygus lucorum, and it contains a1050bp nucleotide sequence encoding350amino acids. The obtained sequence was very closely related to the sequence of lygus hesperus PG by sharing93%sequence identity. AL-PG protein encoded by this gene has a hydrophobic N-terminal signal sequence (MKFTIYALGLLVAVAS A).The calculated molecular mass of the mature protein is35.9KDa with an estimated pI of8.89. The secondary structure of AL-PG consists of many β-sheets, β-turns and random coils, however, there is a small amount of a-helices. The tertiary structure of AL-PG has a right-handed parallel β-helical structure, consisting of four β-sheets, PB1, PB2a, PB2b and PB3. The catalytic amino acid residues, Asp153, Asp174and Asp175are at the bottom of substrate binding cleft. The smallest distance at the entry of the substrate binding cleft is8.4A. Our study also demonstrated that polygalacturonase from Apolygus lucorum could not be expressed in Pichia pastoris expression system.2. Recombinant preparation and inhibiting mechanism analysis of Aspartate aminotransferaseAspartate transaminase from cytoplasm of human cells could be expressed in souble form in Escherichia coli cells. The protein was purified by using HisTrapTM FF crude column chromatography and HiTrapTM Q XL column anion exchange chromatography. SDS-PAGE results showed that the molecular weight of recombinant Aspartate transaminase was about43KDa, and the overall recovery of purified protein obtained was18.3%. Purified protein has very good activity, and it was relatively stable at temperatures25～50℃and pH3.0～10.0. The recombinant protein had optimal activity at45℃and pH9.0. Through inhibitor screening, it was found that aminoxyacetic acid strongly inhibited the activity of this enzyme, the value of IC50was2.16μM. Docking results showed that amino acid residues Trpl41, His190, Ala193, Asp223, Ser224and Arg225formed the activity pocket of the enzyme. Aminoxyacetic acid could form hydrogen bond with amino acid residues His190, Ala193, Ser224and Ala225, and it could tightly bind to the activity pocket of the enzyme. Therefore, it could competitively inhibit the binding between enzyme and its substrate and showed strongly inhibiting effect.