The Loop In Human Lysozyme Scaffold Was Engineered To Be The Active Sites Of Enzyme

The creation of proteins that have new activities is an important goal in protein engineering in which the active site engineering of enzyme plays an important part. An exciting prospect of protein engineering is to engineer new proteins (enzymes) having anticipatory fold or some special function. However, we know a little about the relation between protein structure and function. So it is very difficult to design new proteins (enzymes) possessing new functions according to our desire. Directed evolution is most successful method in protein engineering, which was limited by large scale selection. With the rapid development of protein structure database, the melioration and design of enzyme active site have taken the place of the modification of protein scaffolds used to study engineering enzyme now. Several methods have been established for engineering active site, for example,"active site template"method,"active site graft"method,"active site evolution"method,"active site mimicry"method,"loop graft"method, and so on.We took human lysozyme as a model and obtained"two active-site"enzyme or"bi-function"enzyme through substituting catalytic residues into the loop of human lysozyme. Human lysozyme (EC 3.2.1.17) is an important enzyme used as an anti-inflammatory drug. The human lysozyme gene contains four exons. Exons 1 and 4, code for the amino- and carboxyl-termini, respectively. The amino- and carboxyl-termini increase enzyme stability but do not directly participate in the catalysis. Exon 3 encodes amino acids 82 through 108, Which provide additional substrate specificity and improve catalytic efficiency. Exon 2, encodes amino acids 28 through 82, which may be considered as the original glucosidase, and contains the crucial active site amino acids (Glu35 and Asp53) and a cluster of binding sites for oligosaccharide substrates In the secondary structure of lysozyme, there is a large loop (Cys65 to Cys81), which have no typical secondary structure such as anα-helix or aβ-strand, just a random coil. According to the theory that enzyme active site needs flexibility and loop is flexible, considering crucial amino acid residues in human lysozyme have catalysis, we obtain"two active-site"lysozyme TAL through"exon grafting"method, a"bi-functional"enzyme HLY-RD through"loop grafting"method, and"two active-site"lysozyme NL₂ through substituting catalytic amino acid residues into the loop of human lysozyme.1. Exon grafting yields a"two active-site"lysozyme TALWe grafted an entire exon 2 into the loop of human lysozyme. The target gene TAL was ligated into vector pPIC9 and introduced into Pichia pastoris GS115 (his4). Target protein was expressed in soluble style. The activities of TAL were increased about 2.1 folds. The thermostability and pH stability of TAL were similar to those of natural enzyme. Site-directed mutagenesis were used to prove that TAL possess indeed two active site. Firstly two mutagenic primers were used to convert Glu35 (GAA) and Asp53 (GAC) codons, which are located at the original exon 2 of TAL, to Ala (GCT) codon. Then the mutations happened on the grafted exon 2 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 controls are wild-type lysozyme and the mutants of all of active sites. Activity change and the stereodrawing of TAL simulated through computer showed that TAL really have two active sites.2. A"bi-functional"enzyme HLY-RD was constructed through loop grafting in vitroWe make HLY-RD through grafting the gene of PRD exhibiting RNase-like activity into the loop of human lysozyme. The target gene was 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 31% of the total cell proteins. Inclusion body were washed, sonicated, denatured, then renatured and purified. The purified protein showed a single band on analysis by SDS-PAGE. The final yield is about 320mg/L. Activity assay and stability assay of HLY-RD were carried out and the results showed that HLY-RD contained about 78% activity of lysozyme, whilst the RNase-like activities of HLY-RD was about 150% of PRD. The stability of HLY-RD for lysozyme activity was close to that of wild-type lysozyme. Theλmax of HLY-RD fluorescence emission spectra was found to be 345nm, which is same as wild-type lysozyme. The intensity is higher. Grafted PRD had no big effect on the Tyr63 and Trp64 which located on the substrate combination cleft of human lysozyme. The results of CD spectra and structure simulation showed that two smallα-helix formed in the secondary structure of grafted PRD. So the structure stability of grafted PRD is increased and the binding of HLY-RD and substrate RNA became stronger. This make its activity increasing.3. Substituting catalytic residues into the loop to increase activity of human lysozymeThe mutations were projected on the basis of spacial distance and outspread direction of side-chain between Glu35 and Asp53. In wild-type human lysozyme, the distance between the Cδof Glu35 and Cγof Asp53 is 7.2 ?. Two rational designs were done with the aid of protein three-dimensional structure analysis software SPDBV and PYMOL. One is Asp67 is kept as one of key active sites, Ala73 is mutated to Glu as another one. In NL1, the corresponding distance is 7.23 ? which is similar to wild-type lysozyme. The prolongation direction of Asp67 and Glu73's side-chains are same and point to the interior of protein structure. Another plan is Gly68 is mutated to Glu and Ala73 is mutated to Asp as active sites. In NL₂, the corresponding distance is 7.17 ? which is similar to wild-type lysozyme. The prolongation direction of Glu68 and Asp73's side-chains are same and point to the exterior of protein structure.In order to confirm if NL₂ exhibited lytic activity when the corresponding amino acid residues in the loop of human lysozyme were mutated, we construct a mutation (Glu35Ala/Asp53Ala) of human lysozyme which lost lytic activity. On the basis of it we mutated the corresponding amino acid residues in the loop to catalytic residues to carry out NLM1 and NLM2. They were expressed, purified and assayed their activities. The results suggested NLM1 had no lytic activity but NLM2 exhibited 86.7% activity of wild-type lysozyme, and may be form new active sites."Two active-site"lysozyme NL₂ were constructed using protein three-dimensional structure analysis software SPDBV and PYMOL. The target gene was 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 28% of the total cell proteins. Inclusion body were washed, sonicated, denatured and renatured. 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. The final yield is about 300mg/L.Activity assay of"two active-site"lysozyme NL₂ were carried out and the results showed that the relative activity of NL₂ assayed was 175.9% of WL. The fluorescence emission spectra exhibited that the emission maximum of NL₂ was found to be 345nm, which is same to the wild-type lysozyme. Enzymes+substrance increased the fluorescence intensity atλmax wavelength, but there were some contrast existing in them, which is related to the influence by substrate on Tyr63 and Trp64 which joints the bottom substance and works together with Glu35 to hydrolyzes theβ-1-4 glycosidic bond. The results of CD spectra and structure simulation demonstrated that there was no big difference between"two active-site"lysozyme and wild-type lysozyme. However, we must adopt the further research to describe their structures.In any case, we acquire"two active-site"lysozyme TAL,"bi-functional"enzyme HLY-RD and"two active-site"lysozyme NL₂. This is likely to provide a possible model for engineering"multi-function"enzymes or"multi active-site"enzymes, but also offer practical engineering enzyme.