The Enzyme Modification Based On Fusing With Self-Assembling Amphipathic Peptides

Self-assembling amphipathic peptides?SAP?are short peptides constituted by alternating hydrophobic and hydrophilic residues,which could self-assemble into the nano-particles in solution.Previously studies showed that SAP could influence the expression,thermal stability and enzymatic properties of lipoxygenase?LOX?when fused it to the N-terminus of the protein.To improve the efficiency of SAP,first,we studied the factors that might be involved in SAP-mediated protein production and thermal stability in in Escherichia coli expression platform.The S1?AEAEAKAK?₂ from the Zuotin protein in Saccharomyces cerevisiae was selected as the original SAP.Based on these key factors analysis,two multifunctional tags,a library of expression tags and a thermalstabilization strartegy based on SAP were proposed.Furthermore,we applied the SAP in the metabolism regulation to modify the metabolic enzymes function.The main results achieved in this study are highlighted as follows:?1?The key influential factors on the expression and thermal stability of the SAP fusionsTo understand the influence of SAP composition on the protein expression,S1 and its variants were fused to the N-terminus of green fluorescent protein?GFP?individually.The fluorescence intensity detection results indicated that the change of hydrophobicity?GRAVY,-2.65⁰.4?and net charge?+20^-20?made the fluorescence intensity fluctuate-0.98⁷.22 and2.6¹2.7-fold higher than wild-type GFP,respectively.The length?1/2⁹ units of S1?and hydrophilic residues?with Glu changed by Asp and Lys replaced by Arg and His?made the fluorescence intensity fluctuate 6.34¹0.15 and 6.4¹0.2-fold higher than wild-type GFP,respectively.Thus,the key factors that influenced the expression quantities of SAP fusions were indentified as the hydrophobicity and net charge of SAP.To investigate how SAP composition influences the enzymatic thermal stabilities,S1 and its variants were fused to the N-terminus of polygalacturonate lyase?PGL?individually via PT-linker?PTPPTTPTPPTTPTPTP?,yielding the PGL fusions with different SAP.The thermal stability detection results indicated that the length and charge distribution made the half-lives of PGL fusions fluctuate 0.81².67,1.15¹.8-fold higher than that of PGL,respectively.The linkers with different length or flexibility were inserted into the S1 and PGL,yielding the fusions with different linkers.The thermal stability detection results showed that the half-lives of these PGL fusions fluctuated 0.44².82-fold higher than the wild-type PGL.Thus,the SAP length,linker length and flexibility were indentified as the key factors influencing the thermal stabilities of SAP fusions.?2?Design of multi-functional tags based on SAPFour lysine residues in S1 were mutated into histidine residues,yielding the S1cv2?AEAEAHAH?₂.After fusing S1cv2 at the N-terminus of PGL,LOX and GFP through PT-linker,the expressions of the three fusions in E.coli BL21?DE3?were enhanced by 3.8,0.2 and 1.52-fold,respectively,compared to the corresponding wild-type protein.Moreover,the three S1cv2 fusions were purified with high purities and acceptable recovery rates?47%,39.15%and 56%?due to their affinity to the nickel column.In addition,S1cv2 fusions induced the half-lives of PGL and LOX exhibiting 1.3 and 3.1-fold enhancement,compared to the corresponding wild-type,respectively.The frequently used His-tag with PT-linker had negligible effects on expression.And nickel column affinity purification yields of the three His-tag fuisons were less than 9%.The S1nv12?ANANARAR?₄ could enhance the protein expression more efficiently than that of S1cv2.Four histidine residues were separately inserted into the S1nv12?ANANARAR?₄,yielding the multifunctional tag,S1vw?HNANARARHNANARARHNANARARHNARARAR?.After fusing S1vw at the GFP N-terminus through PT-linker,the expression of GFP fusion in E.coli BL21?DE3?were enhanced 4.22-fold,compared to the wild-type GFP.And the recovery rates of S1vw fusion purified by nickel column was 64.35%.Moreover,the expression of GFP fusion was enhanced 1.91-fold in Bacillus subtilis,while the production of GFP fusion changed slightly in Pichia pastoris.?3?An efficient expression tag library based on SAPBased on the factors that might be involved in SAP-mediated protein production,an expression tag library composed of SAP,which varied in positive net charge?+1⁺20?,was constructed based on the random amplification of S1nv1?ANANARAR?₁₀.The efficiency of the library was validated by PGL,LOX,L-asparaginase?ASN?,and transglutaminase?MTG?.To accelerate preliminary screening,each target enzyme was fused GFP at the C-terminus as the fluorescent screening marker.Sequence analysis results indicated that the SAPs with+2⁺6 net charges were optimal for protein expression enhancement.Finally,application of the library improved the expression of PGL,LOX,ASN,and MTG by 8.3,3.5,2.64 and3.68-fold relative to that of the corresponding wild-type,respectively.?4?A thermostabilization library based on SAPBiochemical analysis demonstrated that SAP could induce protein oligomerization via intermolecular hydrophobic interactions.The size and intensity of the oligomerization could be adjusted by the composition of SAP and linker.Based on this mechanism,a comprehensive protein stabilization strategy was proposed,in which a library of stabilizing tags through random combination of different SAP and linker peptides was developed to design the fusion composition.Application of the library improved the half-lives of PGL,LOX and ASN by5.98,4.96 and 2.95-fold relative to that of the corresponding wild-type,respectively.And the positive mutation rate of the three libraries were 35.8%,21.8%and 25%,respectively.Moreover,the sodium chloride?NaCl?was used to enhance the intermolecular hydrophobic interactions of SAP fusions.After 2 M NaCl addition,the half-lives of the wild-type PGL,LOX and ASN exhibited 3.2,0.95 and 1.7-fold increases,respectively.While the SAP fusions of the three enzymes were enhanced 4.1,2.2 and 3.2-fold,respectively.These results indicated that the SAP could effectively improve the enzymatic thermal stability and enhance the stabilization effect of NaCl.?5?A metabolite enzymes modification strategy based on fusion of SAPA metabolite enzymes modification strategy based on SAP fusions was proposed.In this strategy,the S1nv10?ANANARAR?₂ was separately fused to the N-terminus of metabolite enzymes.Then the hydrophobicity,length and net charge of SAP in the fusions were changed through the PCR.Based on these variations,the expressions,thermal stabilities and enzymatic activities of the enzymes were regulated.After modification by this strategy,the production yield of?-carotene was improved 3.91-fold than that of the original strain.Western-blot analysis indicated that the expression quantities of geranylgeranyl pyrophosphate synthase?CrtE?,phytoene dehydrogenase?CrtI?and lycopene cyclase?CrtY?were changed after SAP fusing.The catalytic efficiency and half-life of the CrtY were improved 0.89 and 1.23-fold than that of wild-type,respectively.As proof of concept,the biosynthetic pathways of eriodictyol and?2S?-naringenin were regulated with the target products exhibiting 1.2 and1.86-fold increase than that of the corresponding original strain,respectively.