Table 1.
Genetically modified proteins in nature and engineered
| Protein | Modification | Structure characteristic | Function | Application |
|---|---|---|---|---|
| Rat peroxisomal enzyme rpMFE16 | Quinque-functional hybrid enzyme (Nature) | N-terminal active site: hydratase domain (A) – flexible linker – isomerase domain (B) – positively charged tunnel – C-terminal active site: (3S)-hydroxyacyl-CoA dehydrogenase HAD superfamily domains (C-E) fusion | Δ3,Δ2-Enoyl-CoA isomerase, Δ2-enoyl-CoA hydratase-1, (3S)-hydroxyacyl-CoA dehydrogenase activities | Enhancing metabolic efficiency in β-oxidation pathway of fatty acid degradation |
| Human intestinal bacterium Ruminococcus gvanus E1 α-galactosidase, AgaSK3 | Bifunctional hybrid enzyme (Nature) | N-terminal GH36 α-galactosidase domain (residues 1-720) – C-terminal nucleotide-binding motif (residues 721-935) | α-Galactosidase and sucrose kinase activities | Enhancing metabolic efficiency: melibiose and raffinose hydrolysis and phosphorylation of sucrose on the C6 position of glucose in the presence of ATP |
| Membrane protein, COX-2-10aa-mPGES-12 | Bifunctional hybrid enzyme (Nature) | C-terminus of cyclooxygenase isoform-2 (COX-2) - transmembrane linker – N-terminus of microsomal prostaglandin E2 synthase-1 (mPGES-1) | Cyclooxygenase and prostaglandin E2 synthase-1 activities | Acceleration of inflammation and cancers response: coupling reaction of converting arachidonic acid into prostaglandin E2 |
| Hepatitis C protein 3/4A, HCV NS3/4A5 | Bifunctional hybrid enzyme (Nature) | N-terminal chymotrypsin-like serine protease (residues 1-181) – linker residues 184-186 (regulatory switching domain–domain interface) – C-terminal superfamily 2 DExD-box helicase domain | Protease and 3′-5′ helicase activities | Enhancing viral propagation efficiency: duplex DNA and RNA unwinding, viral polyprotein cleavage for viral maturation, the cleavage of host innate immune response antiviral signaling protein (MAVS) and interferon-β (TRIF), autoregulation of the protease and helicase activities |
| Sponge Geodia cydonium galectin1 | Chimeric lectin (Nature) | Homologous sequences recombination within carbohydrate recognition domain and extension of galectin-3-like carbohydrate-binding subsite | Carbohydrate-binding activity | Extension of ligand specificity: accommodation to binding tetrasaccharide terminal GalNAcα1-3 moieties |
| Human dual-specificity phosphatase, DSP8 | Mutant (Nature) | Amino acid substitution L192A and noncatalytic C-terminal extension (residues 206-330) | Protein phosphatase and alkaline phosphatase activities | Enhancing signaling transduction efficiency: dephosphorylation of phosphoprotein monoesters and β-phosphate of RNA for cancer, diabetes, inflammation, and Alzheimer's disease prevention |
| Thermophilic bacterium G. stearothermophilus α-galactosidases, AgaA4 | Mutant (Nature) | Amino acid substitution A335E, displacement of Trp336 at catalytic subsite | α-Galactosidase and transglycosylation activities | Increasing yield of sucrose: raffinose hydrolysis and enzymatic synthesis of disaccharides |
| Bacteriophage lysine, Ply187N-V12C70 | Chimeric protein (Engineered) | Catalytic domain of Ply187 lysin; bacterial cell-binding domain of PlyV12 lysin (residues 146-314) | Lytic activity | Enhancing antimicrobial therapy efficiency: extension of lytic activity against human pathogens (staphylococci, streptococci, enterococci) |
| Bacillus megaterium steroid hydroxylase, CYP106A265 | Single mutant (Engineered) | Amino acid substitutions A395L or G397P | Cytochrome P450 activity | Enhancing steroid hydroxylation efficiency: extension of regiospecificity to 3-oxo-Δ4-steroids (progesterone, from 15 to 11-position; increase of 11α-, 9α- and 6α-hydroxylation activities up to fourfold |
| Candida antarctica lipaseB, CalB68 | Single, double, triple and quadruple mutants (Engineered) | Amino acid substitutions G39A, T103G, W104F, A141Q, I189Y, L278A | Lipase and amidase activities | Introducing and enhancing amidase activity up to 3-fold wild type activity for the preparation of optically active amines |
| Mannan-binding holothurian A. japonicus lectin, CmAP/MBL-AJ44 | Bifunctional hybrid protein, double mutant lectin (Engineered) | N-terminal alkaline phosphatase domain – flexible linker (G4S)3 – C-terminal mannan-binding lectin domain; amino acid substitutions A137N/F159K in the lectin active center | Alkaline phosphatase and carbohydrate-binding activities | Enhancing lectin activity by 25±5% and utility in enzyme linked lectin assay (ELLA) for early differentiated cervical cancer diagnosis |
| Trametes multicolor pyranose 2-oxidase, TmP2Ox66 | Double mutant (Engineered) | Amino acid substitutions V546C/E542K | D-glucose and D-galactose oxidase activity, mediated electron transfer (MET) | Enhancing catalysis efficiency: carbohydrate biotransformations in food applications (ketose sugar) or as the anodic bio-component in sensors and biofuel cells |
| Paenibacillus macerans cyclodextrin glycosyltransferase69 | Triple mutant (Engineered) | Amino acid substitutions Y260R/Q265K/Y195S | Transglycosidase activity | Enhancing maltodextrin specificity by 60% for production of vitamin C nonreducible highly antioxidant derivative AA-2G |
| Mucor hiemalis endo-β-N-acetylglucosaminidase, Endo-M67 | Single mutant (Engineered) | Amino acid substitution N175Q | Glycosid hydrolyse and transglycosidase activities | Enhancing glycosynthase activity with oxazoline and natural N-glycan for syntheses of glycopeptides/glycoproteins |
| Bacillus subtilis aminopeptidase, BSAP74 | Single mutants (Engineered) | Amino acid substitutions I387A, I387C and I387S | Aminopeptidase activities | Changing substrate specificity for ability to hydrolyze phenylalanine derivatives with large side chains |
| Guanylate kinase (GK)71 | Single mutant (Engineered) | Amino acid substitution S68P | Phosphotransferase ATP-GMP activity | Changing function from enzymatic to protein-binding (GKdom) of membrane associate guanylate kinases (MAGUK) in mitotic spindle orientation and cell adhesion |
| Transmembrane zinc metallopeptidase Neprilysin, NEP75 | Double mutant (Engineered) | Amino acid substitutions G399V/G714K | Peptidase activity | Changing protein cleavage site specificity for cleavage of amyloid β 1-40 (Ab1–40) at Phe20-Ala21 for the treatment of Alzheimer's disease |
| Plant Madagascar periwinkle flavin-dependent halogenase, RebH72 | Single mutant (Engineered) | Amino acid substitution Y455W | Tryptophan-specific halogenase activity | Changing substrate specificity to install chlorine preferentially onto tryptamine for production of halogenated alkaloid |
| Glucose 1-dehydrogenase IV, BmGlcDH-IV73 | Single mutant (Engineered) | Amino acid substitution G259A | D-glucose, D-xylose, D-mannose and D-galactose 1-dehydrogenase activities | Changing substrate specificity to only D-glucose for clinical assay to examine blood glucose levels |
| Bacillus subtilis phytase82 | Bifunctional hybrid enzyme (Engineered) | N-terminal β-1,4 endoglucanase domain – C-terminal phytase domain | β-1,4 endoglucanase and phytase activities | Enhancing nutrition uptake efficiency as a potential feed additive for monogastric animals |
| Xylanase Cel5A–XylT84 Glucanase XylT–Cel5A84 | Bifunctional hybrid enzymes (Engineered) | N-terminal xylonase domain – C-terminal glucanase domain; N-terminal glucanase domain – C-terminal xylonase domain | Heat-active endoglucanase and endoxylanase activities | Enhancing efficiency of β-glucan and beechwood xylan hydrolysis in biorefineries based on plant waste |
| α-Glucanohydrolase, mut-dexA85 | Bifunctional hybrid enzyme (Engineered) | N-terminal mutanase domain – C-terminal dextranase domain | α-1,3-glucanase and α-1,6-glucanase activity | Enhancing efficiency of α-glucans hydrolysis in the prevention of dental biofilm formation |
| Bacillus subtilis laccase, CotA-BglS83 | Bifunctional chimeric enzyme (Engineered) | Insertion of β-1,3;1,4-glucanase gene bglS (2–214 bp) into cotA gene of laccase (between 22–24 bp), forming inter-domain contacts in Gly319-Gly320, Asn354-Ala359 and Leu634-Ser638 loops of CotA-BglS | Benzenediol:oxygen oxidoreductase and β-1,3–1,4-glucanases activities | Enhancing efficiency of hydrolysis of plant cell wall β-glucans and oxidation of aromatic compounds for sugar release from milled sugarcane bagasse |
| Renilla reniformis luciferase, Aluc3043 | Bifunctional chimeric protein (Engineered) | Insertion of epitop sequences into N-terminal ALuc23 region (between residues Pro20-Val30) | Bioluminescence | Enhancing efficiency of optical intensity and expending utilities of luciferases for the broad use in bioassays in vivo and in vitro |
| Multivalent antibody scFv80 | Bifunctional hybrid antibody/ribonuclease (4D5 scFv-Bn) - ribonuclease inhibitor (Bs-UCNP) (Engineered) | Humanized monoclonal antibody 4D5 (scFv) (HER2-targeted module)- flexible linker - bacterial ribonuclease barnase (Bn):cysteine-free C40/82A mutant barstar (Bs) – upconversion nanophosphors (UCNPs) | Ribonuclease, HER2-binding, optical (photoluminescence) activities | Enhancing efficiency of malignant tumor detection deep in the living tissue for accurate molecular diagnosis and therapy of tumor diseases |
| Human lectin L-FCN/MBL79 | Chimeric protein (Engineered) | Lectin MBL carbohydrate recognition domain - L-ficolin (L-FCN) collagenous domain (simplified quaternary structure) | Ligand recognition and effector activities | Enhancing cooperativity between carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics for reducing infection by wild type Ebola Virus |
| Tumor-associated antigens MUC1 and HER292 | Biepitop hybrid antigen (Engineered) | MUC1 subunit – HER2 subunit | Binding antibodies | Enhancing efficiency of breast cancer detection in ELISA for detection of antibodies against MUC1 or HER2 in human serum |
| Enterotoxigenic E. coli colonization factor antigens (CFAs)93 | Multiepitop multifunctional hybrid protein (Engineered) | Seven E. coli CFAs – labile toxin subunit B – binding subunit of heat-labile toxoid (LT) – heat-stable toxoid (ST) | Recognition, binding and toxic activities | Enhancing efficiency of protection against enterotoxigenic E. coli (ETEC) strains in bacterial diarrhea therapy, inducion anti-CFA and antitoxin immunity |