DNA Shuffling Of Snake PLI?s And Screening Of High Activity Mutants

Snake venom phospholipase A₂?svPLA₂?is a major toxic component of snake venoms,which has neurotoxicity,myotoxicity and hemorrhagic toxicity,etc.It can also cause severe inflammatory reactions in envenomed victims.Many snake sera are naturally resistance to snake venom,due to the existence of inhibitors of svPLA₂?PLI?.Among the three types of PLI?PLI?,PLI?and PLI??,PLI?is widely distributed and extensively investigated both in venomous and non-venomous snake species.However,the inhibitory effect of PLI?from different snake species varies greatly,and the underlying reason remains poorly understood.Current investigations on the snake PLI?mainly focus on its separation and purification from snake blood and its activity determination.Little has been done on the structure-function interpretation.The project recombined novel PLI?s using Sinonatrix annularis?Sa PLI??and Elaphe carinata?EcPLI??as the parents by DNA shuffling.Phage display technology was applied to screen highly-active recombinant PLI?.Objective:DNA shuffling based directed evolution strategy and phage surface display methods were utilized for chimera construction and bio-panning of PLI?mutants with higher activity.The relation of amino acid sequence variation,secondary structure alteration and the activity change of recombinant PLI?s were further discussed.Method:?1?Total RNA was isolated from fresh livers of Sinonatrix annularis and Elaphe carinata by using Trizol reagent.The cDNA was obtained by reverse transcription.PLI?from the two parents was cloned using a degenerate primer pair,followed by sequencing.?2?The two parental PLI?s were mixed and digested by DNase I at 16?for 5minutes.The digested fragments with random length were separated on 2%agarose gel,DNA fractions of 50-100 bp was recovered using DNA recovery kit.These PLI?fragments ware firtly ligated by PCR without extra primer,and then chimera PLI?s?cPLI?s?were cloned by PCR in the presence of PLI?primers which contains Not I and Sfi I restriction sites respectively.The cPLI?genes were cleaved by the two enzymes above and ligated to the same double-digested pCANTAB5e phagemid vector.The recombinant plasmids were then transformed into component Escherichia coli TG1 to get the cPLI?bacterial library,which was further infected with M13K07helper phage to generate a phage library,cPLI?proteins would be expressed on the surface of phage coat.The first-round screening of cPLI?was performed in an immune tube coated with 400?g crude venom of the Deinagkistrodon acutus?D.acutus?.The second-round panning was further conducted using 200?g venom of the D.acutus,and the final resulted cPLI?strains with high-affinity were sequenced.?3?For cPLI?expression by pET28c vector,c PLI?genes with high affinity were cloned by PCR in the presence of PLI?primers containing Nde I and Xho I restriction sites.By regular gene engineering protocol,cPLI?was inserted into the pET28c plasmid and transferred into E.coli BL21.The culture conditions,including temperature and time were simply optimized to enhance the yield of cPLI?protein.After inducible expression by IPTG,the bacteria were collected by centrifugation and homogenized by ultrasonication in ice-bath.The supernatant was obtained from lysate by centrifugation and analyzed by SDS-PAGE.The supernatant was also subjected to filtration using a 0.45?m filter and separation on Ni-resin affinity column using elution buffer containing imidazole to purify cPLI?proteins.The anti-hemorrhagic activity of cPLI?was determined by subcutaneous injection of premixed cPLI??90?g?and D.acutus venom?30?g?on Kunming mice.The resulted bleeding spot was quantified using Image-Pro Plus software and compared to the parent controls,named,saPLI?and ecPLI?groups.?4?The amino acid sequence of cPLI?s was obtained based on DNA sequencing.Alignment of cPLI?s and the parental PLI?s was run using DNAMAN software to reveal the reorganization of PLI?.The secondary structure of PLI?s was analyzed using the online tool PSIPRED?http://bioinf.cs.ucl.ac.uk/psipred/?to predict probable regional changes.Homologous modeling of cPLI?and parental PLI?was performed using the online swiss model tool?http://www.swissmodel.expasy.org?.The tertiary structure of PLI?s was predicted and aligned using UCSF Chimera software.Collectively,the relationship between the structure and function of PLI?was discussed.Result:As mentioned above,three chimeric PLI?s,named cPLI?1?C1?,cPLI?2?C2?and cPLI?3?C3?.with high affinity to snake venom PLA₂ were finally obtained by sequential gene cloning,DNA shuffling and phage display bio-panning.Recombinant pET28c-cPLI?plasmids were constructed and transformed into BL21strain.The three types of cPLI??fused with His tag?proteins were expressed in conditions of 22°C,210 rpm shaking and 0.4 mM IPTG induction.c PLI?was purified by one-step Ni resin chromatography,evidenced by the single band on SDS-PAGE gel.All the three cPLI?showed good anti-hemorrhagic effect on D.acutus venoms,of which the C2 was the strongest one even compared to the parental PLI?s.The saPLI?,EcPLI?and the three cPLI?s shared 91.48%identity in amino acid sequence.Their secondary structure consist of similar?-helix,?-turn,and random coils.The amino acids proportion of?-turn in there five PLI?s is 31.87%-34.64%,of which SaPLI?and cPLI?1 is the lowest and the highest respectively.?-helix lies mainly at the C-terminus of the peptide,whose proportion was 1.1%in C1 and C3,1.6%in EcPLI?,3.28%in C2,and 3.3%in saPLI?.Besides,the length of 150-155?-helix was also different,such as C1 and C3 only contains 2 amino acids?150-151Aa?,ecPLI?contains 3 Aa(150-152,while saPLI?and C2 are formed by 6Aa?150-155?.The length and position of?-helix may be related to the activity of PLI?.Conclusion:The DNA shuffling and panning techniques were successfully established.Three chimera PLI?s were obtained using SaPLI?and EcPLI?as parents.All these three types of cPLI?showed good anti-hemorrhagic activity against D.acutus venoms after expression by pET28c vector,of which C2 was the best one.Structural analysis indicated the peptide RPFPGLPLSHPNGYI?85-99?might be an effective segment of PLI?.There was slight difference in the secondary structure of parental PLI?s and cPLI?s,For instance,C2?the isoforms with higher activity?and SaPLI?compose of a six-amino acid?-helix from 150-155,while the length of?-helix in other types of PLI?is less than 3 amino acids.PLI?from Bungarus multicinctus was found lack of?-helix at the same position?150-155aa?,which has little anti-hemorrhagic activity.Therefore,?-helix at this specific position and its length might affect the activity of PLI?greatly.Moreover,the number and position of?-sheet in PLI?may also relate to the its activity in snake venom neutralization.However,these bio-informative analysis need further experimental validation.