Codon Optimization And Prokaryotic Expression Of Capparis Masaikai Sweet Protein Gene(Mabinlin?) In Escherichia Coli

Plant sweet protein was a plant protein isolated from plant species.Due to its high sweetness,low calorie,safer than synthetic sweetener,safety and non-toxicity,sweet proteins was widely favored as a new type of sweetener.The main objective of this study was to explore the expression of MabinlinII sweet protein in prokaryotes and detect the sweetness of the expressed protein.The plants were mainly distributed in tropical and subtropical regions such as Yunnan and Guangxi in China.The number of Capparis Masaikai plants were scarce and endangered because of the natural factors such as geography and climate.In this study,the expression of the optimized Mabinlin? sweet protein gene by means of an E.coli expression vector,it could be used to investigate whether the sweet protein could be expressed in large amounts.Provide some theoretical basis for the development of Mabinlin? sweet protein.At present,the Mabinlin? sweet protein gene has been expressed in E.coli.In order to further increase the expression level and determine its sweet taste activity,the full-length sequence of the Mabinlin? sweet protein gene was obtained by NCBI query.Then the codon was replaced with a common codon and the 468 bp length transposase codon was optimized on the basis of the codon preference and degeneracy of E.coli.The purpose of this study was to use pET28a?+?as a plasmid vector and E.coli BL21?DE3?as a host strain to express the codon-optimized 468 bp length MabinlinII Plus sweet protein gene,and the 321 bp length MabinlinII?AB?Plus sweet protein gene without signal peptide,N-terminal peptide,C-terminal peptide.Compares the two sequences which could express the protein better,and studied how to increase the protein expression.In order to obtained more efficient expression,this research also has carried on the express condition optimization.Western Blot was used to identify the expressed protein,and Ni-NTA agarose resin was used to purify the obtained protein.Finally,sensory evaluation experiment and electronic tongue test were used to determine the sweetness of the target protein.This experiment was mainly divided into four parts.The first part was the construction of recombinant expression vector.Firstly,the codons of Mabinlin? sweet protein gene coding region sequence?GI:1817545?weree optimized according to the degeneracy and preference of E.coli.The optimized Mabinlin? sweet protein gene is named as MabinlinII Plus.Removed the signal peptide,N-terminal peptide,and C-terminal peptide of the MabinlinII Plus sequence,the new gene after the cleavage was named as MabinlinII?AB?Plus.The restriction enzyme BamHI and Xho I were used to double-cleave the cloning vector containing the gene sequences MabinlinII Plus/MabinlinII?AB?Plus and the expression vector pET28a?+?respectively.And then using T4 DNA ligase to ligate the expression vector pET28a?+?with the gene sequences MabinlinII Plus and MabinlinII?AB?Plus respectively.The recombinant plasmids pET28a?+?-MabinlinII Plus and pET28a?+?-MabinlinII?AB?Plus were transformed into DH5?competent cells respectively.Culture colony overnight,and then the PCR assay was carried out.0.2 mL of the positive bacterial solution for the electrophoresis result was sent to the sequencing company.BioEdit software was used to compare nucleotide sequences with the original sequence.The results of sequence comparison indicated that the recombinant expression vectors pET28a?+?-MabinlinII Plus and pET28a?+?-MabinlinII?AB?Plus were successfully constructed.The second part was the optimization of prokaryotic expression and expression conditions of the recombinant vectors pET28a?+?-MabinlinII Plus and pET28a?+?-MabinlinII?AB?Plus.In this experiment,BL21?DE3?was selected as the host strain for expression,and the two recombinant vector plasmids were expressed in BL21?DE3?and analyzed by SDS-PAGE?SDS polyacrylamide gel electrophoresis?.The results showed that the recombinant vector pET28a?+?-MabinlinII Plus had a correspondingly less pronounced band at a molecular weight of approximately 26.0 kDa.After optimizing the expression conditions of IPTG concentration,induction temperature,induction time and induced initial growth amount OD₆₀₀,the expression level was not improved.However,the recombinant vector pET28a?+?-MabinlinII?AB?Plus had a significantly induced expression band,and the protein bands were significantly deepened after optimization of expression conditions,the results showed the target protein is highly expressed.The unoptimized recombinant vector BL21?DE3?/pET28a?+?-MabinlinII?AB?and the optimized recombinant vector BL21?DE3?/pET28a?+?-MabinlinII?AB?Plus were induced and expressed under optimal conditions.Total protein was analyzed by SDS-PAGE and Band Scan gel imaging system.The results showed that the expression level of the recombinant vector BL21?DE3?/pET28a?+?-MabinlinII?AB?Plus after optimization was 33%,the expression level of the BL21?DE3?/pET28a?+?-MabinlinII?AB?was 26%,and the expression level of the recombinant vector after gene optimization was7%higher than that before gene optimization.The third part was the purification and identification of the target protein generated by recombinant vector pET28a?+?-MabinlinII?AB?Plus in BL21?DE3?.The total bacterial protein produced by E.coli was analyzed by SDS-PAGE.It was observed that there was a protein band at 19 kDa with expectations.Therefore,the repeated freeze-thaw,ultrasonic broken and centrifugation assay were used to collect the supernatant protein for protein purification.The finally collected purified sample was analyzed and identified by Western Blot.The results showed that the recombinant protein of interest was the genetically optimized Mabinlin? sweet protein.The fourth part was the sweet taste detection of Mabinlin? sweet protein.Two types of sweetness detection methods were selected.The first one was sensory detection.The"A"-non-"A"test was used to assess the taste of the sample.After tasting,the evaluator could accurately identify the sample and state the taste characteristics of the sample.Compared with the sucrose standard solution,the sample had a strong post-sweetness,which could clearly distinguish the sample from the control;the"double-blind"sensory test measures that measured the sample showed that the sweetness of the sweet protein is about 400 times of the 2%standard sucrose solution.The second method was to use a bionic electronic tongue for qualitative and quantitative analysis of samples.The samples were compared with sucrose,sodium chloride,magnesium sulfate,citric acid and sodium glutamate.Determine the sucrose solution with different concentration gradients and made a standard curve.Quantitative analysis results showed that the sweetness of Mabinlin? sweet protein produced by 100 mL E.coli was equivalent to the sweetness of0.4 g sucrose with a purity of 99.7%.