Construction Of Several Highly Efficient Expression Vectors And Modification Of Glucanase PfLamA Based On A Metagenomic Segment Shuffling Method

Molecular cloning is a fundamentally important step in biotechnology,genetics,pharmaceuticals,protein biochemistry,structural biology,etc.In recent years,with the rapid development and deployment of next-generation sequencing technologies,huge amounts of genomic information are being generated.To analyze and utilize such important genomic information in functional studies,efficient tools for molecular cloning and protein expression are necessary.Existing cloning and expression vectors still need further improvement for more efficient cloning,expression and protein purification.Therefore,in this study a series of novel plasmid vectors have been designed and constructed,which provided important tools for solving the above problems.In this study,the novel constructed plasmids were used for molecular cloning and expression of endoxylanase genes of pfLamA from Pyrococcus furiosus and Hj-Xyn from Trichoderma reesei.At the same time,using the novel vectors,the pfLamA was modified through a novel metagenome-based DNA shuffling strategy.1.The existing pET vector cloning efficiency is not high enough,false positives are prone to occur,and the selection of cloning sites is not flexible enough.To solve these problems,in this study we have constructed a novel minitype plasmid vector pANY2.This vector uses ccdB negative selection marker,which can introduce higher cloning efficiency and very low false positive rate.In addition,the multiple cloning sites of the vector can meet the needs of sticky-end cloning,blunt-end cloning and recombinase cloning.More importantly,the multiple cloning sites also contain specially designed AhdI restriction sites that can be used to create T-vectors in the laboratory to perform TA-cloning.In addition,the plasmid also has a T7 promoter and a His-tag,which can meet the need for efficient protein expression and purification.This novel plasmid may find wide range of applications.2.In this study,in order to solve the problem of low cloning efficiency of temperature-induced plasmids,a new plasmid vector pANY3 containing the temperature-sensitive control elements of cIts857-pR/pL has been constructed,which can not only significantly improve the cloning efficiency,but also introduce temperature-induced protein expression.In addition,the effect of induction temperature on protein expression and enzyme activity was further analyzed in this study.The results showed for the first time that the influence of induction temperature on the expression level of different proteins may be very different,so optimization of induction temperature is always necessary.According to the reported studies,fixed temperature of 42 °C is commonly used for induce expression,and the results of this study should be valuable for correcting this inappropriate practice.3.Existing expression vectors can only be expressed unidirectionally,therefore only one target recombinant plasmid can be obtained in a single cloning step.In this study,a novel bidirectional expression plasmid was constructed,with which two target recombinant plasmids can be obtained simultaneously by TA cloning.The insertion of the target gene in the two target recombinant plasmids is reversed.Under different promoters,IPTG-induced expression or temperature-induced expression can be achieved.In this way,two clones can be obtained in parallel,and the expression efficiency of two expression systems for the same gene can also be conveniently compared.This bidirectional expression vector is novel and important for related research.4.The commonly used His-tag-based recombinase purification methods have the disadvantage of high cost.In addition,the use of imidazole often affects the enzyme activity.In this study,a new plasmid vector pANY6 has been constructed,which could not only perform efficient molecular cloning,but also use a simple salt precipitation method to achieve the purification of recombinant proteins.This plasmid contains a fusion tag with intein and elastin-like protein(ELP)with a self-cleavage function.The fusion protein can be separated by a precipitation step using an ELP tag,and the ELP tag can be excised by an intein.In addition,this study also explored the coupling of ELP precipitation purification process with urea denaturation,refolding and purification of inclusion body processes,which can effectively avoid the time-consuming operation of traditional dialysis method to remove urea.Using this simple strategy for pfLamA purification,the final purification concentration was increased nearly 3 times while the total enzyme activity was increased by nearly 50%.The results of this study are particularly important for the purification of insoluble recombinant proteins.5.In this study,with the newly constructed vector,the pfLamA was molecularly modified using the metagenomic segmentation method.The metagenomic 16 s was sequenced to determine the microbial composition of soil samples.Based on this,degenerate primers were designed through the conserved region of pf LamA,DNA fragments homologous to pf LamA were obtained from soil genomes by PCR,and the full-length genes were recombined by overlap extension,and then ligated to the plasmid vector to construct a mutant library for activity screening.This method of metagenomic segment shuffling has not been reported.Therefore,this study has provided a new idea for molecular modification of soil microbial enzymes,and also verified the effectiveness of the newly designed vector.In summary,this study designed and constructed a series of novel plasmid vectors,which have been used for molecular cloning,heterologous expression,protein purification of pfLamA and Hj-Xyn.In addition,modification of protein molecules based on metagenomic reorganization strategies has been realized based on a newly designed plasmid.These new plasmid vectors and their related research methods are very valuable for similar research,which may have wide application in the fields of biological sciences,biotechnology,agriculture,food,etc.