The "Double-centre" Lysozyme Constructed By Exon Reduplication And Rearrangement In Vitro

During past ten years, directed molecular evolution of enzymes is a powerful engine for the creation of new phenotypes and an exciting development happened on the research of enzyme molecular structure and function and applied to the field of medical, industrial and agriculture. Now directed molecular evolution extends from gene and protein molecular to genome and proteome, metabolic pathway and virus, and further to directed molecular directed of cells. Directed evolution of enzyme active centre plays a key role in this field.Active centre engineering of enzyme has been the most active field that biochemists and chemists make the best efforts to engineer enzymes of improved properties and functionalities. Several methods have been established for engineering active centre, for example, "active centre template"method, "active centre graft" method, "active centre evolution"method, "active centre mimicry"method, "loop graft" method, and so on. With mimicking the active centre structure of the L-aspartase (tetramer), we obtained a monomeric L-aspartase by in vitro selection. Considering RNA has higher flexibility than DNA, we have successfully acquired the circular RNA-DNA enzyme by in vitro selection, which are composed of catalytic domain of RNA as the active centre and DNA as backbone. Followed above idea, a novel replicating circular DNAzyme was constructed by cloning the 10-23 DNAzyme gene into M13mp18 vector. Here, we described constructing the "double-centre" lysozyme by exon2 reduplication and rearrangement (codon rearrangement) in vitro.Duplication and rearrangement of the exons would result in novel genes that express proteins with diverse functional modules. We take human lysozyme as a model, acquired the "double-centre" enzyme through exon reduplication or rearrangement. Lysozyme(EC3.2.1.17) was discovered more than eighty years ago in 1922 by Alexander Fleming and a kind of unspecific immunal factor consisting in normal human body liquids and tissues. It was completely no harmful and side-effect to human body, and has the function of antibacterial, anti-inflammatory, anti-viral and anti-tumor. Human lysozyme is an important enzyme used as an anti- inflammatory drug. Lysozyme dimmer appears anti-HIV activity (hydrolyze polysaccharides of virus) obviously. The enzyme, with a higher stability, has long been a good model for the study of the relationship between genes and protein structures. Human lysozyme gene contains four exons. Exonl and 4, coding for the N- and C- terminal regions of the protein, respectively. Those encoding N- and C- terminal regions increase the stability of enzyme, however, do not directly participate in the catalytic function. As well, the additional substrate specificity and improving in catalytic efficiency of the active centre were determined by Exon3, coding for amino acids 82-108. Exon2, coding for amino acids 28-82, which may be considered as the original glucosidase, and containing the amino acid residue of active center (E35 and D53) and a cluster of binding site of oligosaccharide substrate. Between E35 and D53 there is a cleft dividing the lysozyme protein into two main domains. The sugar ring of substrate lies lower down the cleft and then the 8-1-4 glycosidicbond between sugar D and E were cleaved. A large loop, which consists of residues C65 to C81, exposes outside the lysozyme molecule and there dose not exist any typical secondary structure such as an a-helix or a 6-strand in this loop. A restriction site Hindi between codons corresponding to V74 and N75 of lysozyme was selected as convenient for us to construct the vector. V74 and N75 are present in the loop locating between exon2 and exon3 coding regions of lysozyme gene. So in virtue of the restriction site Hindi, we can make exon2 reduplication and rearrangement more easily through inserting exon2 into the loop. Reduplicate exon2 was amplified by PCR and rearranged exon2 (codon rearrangement) was chemically synthesized. Then the "double-centre" lysozyme were constructed through experiment method of restriction endonuclease treatment, ligation and PCR.In this research we selected yeast Pichia pastoris as the host strain. Expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. It has a lot of advantages as expression hosts for the existence of well-established fermentation methods and the presence of methnol-regu lated promoters. Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification. The gene of wild-type human lysozyme(WL), exon2 reduplication lysozyme(RD-L) and exon2 reanangement lysozyme(RA-L) were cloned into expression vector pPIC9, then transformed into Pichia pastoris GS115. The "double-centre" lysozyme were induced by methanol and secreted intothe medium. Some methods were used to optimize expression but the level of protein expression was very low versus 3.8mg/L RD-L, 2.1mg/L RA-L and 4.3mg/L WL, respectively. It's difficulty to purification of enzymes, enzymology character experiment and analysis of structure and function, so we change to select E. coli as host strain.The target genes were ligated into vector pET21a(+) and introduced into E. coli BL21(DE3). Escherichia coli BL21 (DE3) strains harboring the recombinant vectors expressed high-level of target protein in inclusion body, which consisted about 42% (RD-L), 31% (RA-L) and 38% (WL) of the total cell proteins (see Fig. 3A). The yield of wet weight cells was about 6g/L in fermentation culture versus 470mg/L RD-L, 350mg/L RA-L and 440mg/L WL, respectively. The renatured supernatant was applied onto a CM Sepharose (Fast Flow) and Sepharcryl S-100 in tandem. The purified protein showed a single band on analysis by SDS-PAGE. Activity assay and stability assay of the "double-centre" lysozyme were carried out and the results showed that the relative activity of RD-L assayed was about 181.5% of the WL, whilst the activity of RA-L was about 107.2% of WL. The stability of the "double-centre" lysozyme was close to that of wild-type lysozyme.To prove that the "double-centre" lysozyme possess indeed two active centre, four primers were used to do site-directed mutagenesis. Firstly two mutagenic primers were used to convert Glu35 (GAA) and Asp53 (GAC) codons, which are located at the original exon 2 of the "double-centre" lysozyme, to Ala (GCT) codon. Then the mutationshappened on the reduplicate exon2 and rearranged exon2 completed by the same method as described above. The mutation genes were expressed and activity was measured by a lysis using M. lysodeikticus cells as substrates. The control are wild-type lysozyme and the mutants of all of active sites. Activity change and the stereodrawing of the "double-centre" lysozyme simulated through computer showed that RD-L really have two active centres.Lysozymes antibacterial experiment showed that lysozyme had restraining effect on gram positive bacteria, but had no obvious restraining effect on gram negative bacteria. Lysozymes had a little effect on gram negative bacteria with EDTA. Metal ions had some influence upon activity of enzymes, the result was that K+, Na+ > Mg2+, Mn2+ > Ca2+, Zn2+, Co2+ > Cu2+ ? Fe3+, Pb+.Study of the ultraviolet spectrum showed that Trp was active and decisive amino acid. Trp jionts the bottom substance and works together with Glu35 to hydrolyzes the linkage between muramic acid and Nacetylglucosamine of mucopolysaccharides. Restraining substances. Enzymes+substrance changed the ultraviolent absorbing value at special wavelength.The "double-centre" lysozyme enhanced the number of reveal Trp and enhanced the capacity of jionting the bottom substance, increased antibacteria effect.In conclusion, we obtained novel lysozymes which had two active centre undoubtedly by exon2 reduplication. This may serve as an approach engineering the active centre and improving the function of enzymes.