Preparation,Biological Characteristics Of Alpha-amylase Inhibitor CL-AI Protein And Analysis Of Its Spatial Structure In Chickpea (Cicer Arietinum L.)

Alpha-amylase inhibitor(?-AI)is kind of glycosidase inhibitors,which widely exists in the endosperm of plant seeds and microorganisms metabolites.As a new diabetes drug,it functions in hypoglycemic and lipid-lowering by reducing the body's digestion and absorption of dietary carbohydrate and fat production through inbihiting the biological activity of amylase,a key enzyme in the body glucose metabolism.At present,?-AI has been applied popularly in the field of medicine and agriculture,exhibits unique traits in clinically treatment of diabetes,hyperglycemia,hyperlipemia,lipomatosis.The efficacy and safety of the amylase inhibitor depend on the processing and extraction techniques used.A remarkable negative correlation between starch content and inhibitory activity of?-amylase inhibitor were found among different varieties,and the gene sequences of a-amylase inhibitor proteins CL-AI in chickpea were cloned and the full-length ORF of CL-AI gene was 1491 bp,encoding 497 amino acids.On the basis of previous studies,the follow studies were carried out.First,Prokaryotic expression and purification of the chickpea CL-AIgene and CL-AI-?,CL-AI-? Subunits were researched;Second,Research on biological characteristics of recombinant proteins CL-AI were on the agenda;Third,The screening of CL-AI crystallization conditions were studied;Main results of this study are as follows.1.The sequence of CL-AI and CL-Al-?,CL-AI-? were inserted into prokaryotic expression vector pE-SUM03 respectively,and then transformed into competent E.colil Rosetta(DE3).The recombinant protein was highly expressed after induction with 0.6 mmol/L IPTG at 28 ? for 4h,CL-AI,CL-AI-a solube fusion protein and CL-AI-?inclusion fusion protein were obtained though Ni2+ affinity chromatography and CL-AI,CL-AI-a solube fusion protein were further purified via gel-filtration chromatography,Western blotting analysis suggested that CL-AI,CL-AI-? protein had a strong reaction with anti-His-tag antibody,exhibiting as a single clear band,and CL-AI,CL-AI-a were present as monomers,which met requirements for protein crystallization.2.The CL-AI-? gene expression vectors with His-SUM03,MBP,Trx-His,GST,GST-His labels were successfully constructed and used in induced expression in Escherichia coli,when IPTG induction concentration were 0.2 mmoL,induction temperature were 15?.The CL-AI-? recombinant soluble proteins were eventually obtained in pCold-SUMO vector with cold promoter and His-SUM03 double tags.The prokaryotic soluble expression system of CL-AI was established and optimized by constructing different Prokaryotic expression vector with different tags of CL-AI-P protein.Determination of inhibitory activities of prokaryotic expression fusion protein against human salivary amylase(HSA)respectively showed that CL-AI,CL-AI-a,CL-AI-? had inhibitory against HSA,with inhibitory activities were 71.52%,68.56%,63.75%respectively,but there was no significant difference between them(P>0.05).3.The gene sequence of alpha-amylase AAMY1 were isolated from the seeds of the Chickpea,AMY1 inclusion fusion protein were obtained from induced protein expression and using Ni column affinity chromatography and AMY1 inclusion body protein was taken renaturation in vitro.The result of enzyme activity assay showed that CL-AI-a protein had specific inhibitory effect on endogenous alpha amylase AMY1 protein in chickpea,with inhibitory activities were 45.73%.CL-AI protein with amino acid point mutation results showed that His353 mutation affected inhibitory activity of the CL-AI protein,When the content of the mutant protein was 0.07 ?g,the inhibitory effect of the mutation on HSA was significantly decreased with only remain activitity 35.03%.But after GIu453 mutation for Val,CL-AI protein has remain inhibitory activitity(53.25%)on HSA.With the increase of protein content,its inhibitory activitity rose and the inhibitory activity of the CL-AI mutant protein was higher than the control group at high content(0.28 ?g).The effect of CL-AI on seed germination of maize,wheat and rice showed that there was no obvious effect on plant seeds germination rate,but CL-AI could slow down the growth rates of root and shoot length,indicating delayed growth after seeds germination,thus the simplified vigor index of seed decreased obviously,and the difference reached significant level(P<0.05)?4.Crystal screening of CL-AI was carried out with the purified protein obtained aforementioned.By the gas phase diffusion method,the shape of the initial screening crystal were mainly divided into three types:tablets layered or cluster,needle or rice-shaped,cylindrical or polyhedral.The conditions were optimized by using the hanging drop vapor diffusion method,and the results was as follow:20%of PEG2000,0.2 M of MgCl2 and sodium citrate pH 6.5,the concentration of CL-AI was about 10 mg/mL and the crystallization temperature could be 16?,fine crystals were obtained as needle-like.Diffraction data of saved crystals was collected and the refined resolution of CL-AI was 1.9A.