Loop Grafting Yields The "Bi-functional" Enzyme And "Double-centre" Enzyme

The creation of proteins that have new activities is an important longtime goal in protein engineering in which the active site engineering of enzyme plays an important part. An exciting perspective of protein engineering is to construct new proteins (enzymes) having anticipatory fold or some special function. As the natural biological catalyst, enzyme could increase the reaction rate to a great extent and also have certain highly specificity at the same time. Currently,"Enzyme Engineering"stands in a profound bio-revolution atmosphere, biologist make a great progress by improving and modifying enzymes in the characters of original physical and catalytic mode. That comes to the strategies: rational design and directed evolution. Some meaningful and valuable accomplishments could be achieved, but both of the strategies described above have certain restrictions.However, we just know a little about the relationship between protein structure and function, 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. In order to change or modify the modern function of protein, the strategy of grafting is always selected by biologist. The method of active site grafting is based on structure rational design which could be combined with irrational design. After constructing the matching part at protein scaffold, the directed evolution in vitro and screen should be carrying out, then we could acquire the designed product.Lysozyme (EC3.2.1.17) was discovered more than eighty years ago in 1922 by Alexander Fleming and a kind of unspecific immune 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. Exon1 and 4, code 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 theβ-1-4 glycosidic bond 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 anα-helix or aβ-strand in this loop. A restriction site HincII between codons corresponding to V74 and N75 of lysozyme was selected as convenient for us to construct the vector.Our lab has done serial research projects about RNAzyme and DNAzyme for many years. In the earlier work, we report that a 10-23 deoxyribozyme-hammerhead ribozyme targeted theβ-lactamase mRNA in initiation and coding regions. The recombinant phagemid vector carrying the 10-23 deoxyribozyme-hammerhead ribozyme gene sequence were capable of replication, transcription in E.coli cells and showed RNA-cleaving activities of the 10-23 deoxyribozyme, the hammerhead ribozyme at replication and transcription levels. Basing on the amino acids sequence encoded by hammerhead nucleotide sequence, PR, the 13 peptide, was synthesized chemically. Similarly, PD came of 10-23 deoxyribozyme. The catalytic function assay indicates that PR has the single strand RNA-cleaving activity, while PD does not. Obviously, hammerhead ribozyme nucleotide sequence could encode relevant amino acids sequence with certain catalytic function. So, we name this peptide'ribopepzyme'that originates from ribozyme and has the similar catalytic function as ribozyme. In addition, for exploring the essence of ribopepzyme, another two peptides were synthesized. PRD, a 25 amino acids peptide, was composed of PR and PD. P2R, a 26 amino acids peptide, was composed of two PR monomers. Further results demonstrated that both PRD and P2R also have the single strand RNA-cleaving activity with certain cleaving site selectivity.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-sites"lysozyme HLY-E2 through"exon grafting"method, a"bi-functional"enzyme HLY-2R,HLY-RD through"loop grafting"method. We constructed a gene engineering expression vector pET21a(+) with target gene, and then transferred it into E.coli BL21(DE3). Escherichia coli BL21 (DE3) strains harboring the recombinant vectors expressed high-level of target protein in inclusion body. Inclusion body were washed, sonicated, denatured, then renatured and purified. Via chromatography of CM Sepharose (Fast Flow) and Sepharcryl S-100, the purified protein showed a single band on analysis by SDS-PAGE.Comparing the activity data among HLY-RD(HLY-2R), Human lysozyme, and PRD (P2R), HLY-RD and HLY-2R do have two enzyme functions, the lysozyme cleavage activity and RNase-like activity. From stability experiment and fluorescence spectrum, the results of HLY-RD(HLY-2R) have similarity as those of wild type lysozyme. But there are only a few differences between HLY-RD and HLY-2R in the basic parts of wild type lysozyme. In order to find out the structure of peptide, we used two methods structure prediction and CD spectrum. All the results show that there are not any definite secondary structures in peptides and most exhibit random coil. When they were being grafted in lysozyme scaffold, the structures of peptides transformed toβ-strands. It would be in all likelihood that those new structures were formed by the effect of lysozyme scaffold and amino acids around. Activity assay and stability assay of the"double-centre"lysozyme were carried out and the results showed that the relative activity of HLY-E2 assayed was about 211.5% of the WL and the same stability. Whether the enzyme activity of HLY-E2 was highly increased for the formation of two activity centers, we mutated the key amino acids of former exon2 and repeated exon2 in"double-centre"lysozyme, respectively. Completed mutant at two positions would be served as control. The results of activity analyses displayed that the relative activity of Mutant 1 was 94.7 and that of Mutant 2 was 102.1 which had the same activity as of wild type lysozyme. Mutant 3 lost its original activity completely. It could be concluded that not only Glu35, Asp53 of former active sites but Glu82, Asp100 of grafting sequence contribute to the enzyme activity. The"double-centre"exists in the enzyme indeed.Most of naturally occurring proteins have certain loop structures, so it would be meaningful to engineering the existent loop to enzyme active sites. This is likely to provide a possible model for engineering"bi-functional"enzymes or"double-centre"enzymes, but also offer practical engineering enzyme.