DNA Shuffling Of Snake PLIγs 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（PLIα,PLIβand PLIγ）,PLIγis widely distributed and extensively investigated both in venomous and non-venomous snake species.However,the inhibitory effect of PLIγfrom different snake species varies greatly,and the underlying reason remains poorly understood.Current investigations on the snake PLIγmainly 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 PLIγs using Sinonatrix annularis（Sa PLIγ）and Elaphe carinata（EcPLIγ）as the parents by DNA shuffling.Phage display technology was applied to screen highly-active recombinant PLIγ.Objective:DNA shuffling based directed evolution strategy and phage surface display methods were utilized for chimera construction and bio-panning of PLIγmutants with higher activity.The relation of amino acid sequence variation,secondary structure alteration and the activity change of recombinant PLIγs 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.PLIγfrom the two parents was cloned using a degenerate primer pair,followed by sequencing.（2）The two parental PLIγs 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 PLIγfragments ware firtly ligated by PCR without extra primer,and then chimera PLIγs（cPLIγs）were cloned by PCR in the presence of PLIγprimers which contains Not I and Sfi I restriction sites respectively.The cPLIγgenes 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 cPLIγbacterial library,which was further infected with M13K07helper phage to generate a phage library,cPLIγproteins would be expressed on the surface of phage coat.The first-round screening of cPLIγwas performed in an immune tube coated with 400μg crude venom of the Deinagkistrodon acutus（D.acutus）.The second-round panning was further conducted using 200μg venom of the D.acutus,and the final resulted cPLIγstrains with high-affinity were sequenced.（3）For cPLIγexpression by pET28c vector,c PLIγgenes with high affinity were cloned by PCR in the presence of PLIγprimers containing Nde I and Xho I restriction sites.By regular gene engineering protocol,cPLIγwas 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 cPLIγprotein.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 0.45μm filter and separation on Ni-resin affinity column using elution buffer containing imidazole to purify cPLIγproteins.The anti-hemorrhagic activity of cPLIγwas determined by subcutaneous injection of premixed cPLIγ（90μg）and D.acutus venom（30μg）on Kunming mice.The resulted bleeding spot was quantified using Image-Pro Plus software and compared to the parent controls,named,saPLIγand ecPLIγgroups.（4）The amino acid sequence of cPLIγs was obtained based on DNA sequencing.Alignment of cPLIγs and the parental PLIγs was run using DNAMAN software to reveal the reorganization of PLIγ.The secondary structure of PLIγs was analyzed using the online tool PSIPRED（http://bioinf.cs.ucl.ac.uk/psipred/）to predict probable regional changes.Homologous modeling of cPLIγand parental PLIγwas performed using the online swiss model tool（http://www.swissmodel.expasy.org）.The tertiary structure of PLIγs was predicted and aligned using UCSF Chimera software.Collectively,the relationship between the structure and function of PLIγwas discussed.Result:As mentioned above,three chimeric PLIγs,named cPLIγ1（C1）,cPLIγ2（C2）and cPLIγ3（C3）.with high affinity to snake venom PLA₂ were finally obtained by sequential gene cloning,DNA shuffling and phage display bio-panning.Recombinant pET28c-cPLIγplasmids were constructed and transformed into BL21strain.The three types of cPLIγ（fused with His tag）proteins were expressed in conditions of 22°C,210 rpm shaking and 0.4 mM IPTG induction.c PLIγwas purified by one-step Ni resin chromatography,evidenced by the single band on SDS-PAGE gel.All the three cPLIγshowed good anti-hemorrhagic effect on D.acutus venoms,of which the C2 was the strongest one even compared to the parental PLIγs.The saPLIγ,EcPLIγand the three cPLIγs shared 91.48%identity in amino acid sequence.Their secondary structure consist of similarα-helix,β-turn,and random coils.The amino acids proportion ofβ-turn in there five PLIγs is 31.87%-34.64%,of which SaPLIγand cPLIγ1 is the lowest and the highest respectively.α-helix lies mainly at the C-terminus of the peptide,whose proportion was 1.1%in C1 and C3,1.6%in EcPLIγ,3.28%in C2,and 3.3%in saPLIγ.Besides,the length of 150-155α-helix was also different,such as C1 and C3 only contains 2 amino acids（150-151Aa）,ecPLIγcontains 3 Aa(150-152,while saPLIγand C2 are formed by 6Aa（150-155）.The length and position ofα-helix may be related to the activity of PLIγ.Conclusion:The DNA shuffling and panning techniques were successfully established.Three chimera PLIγs were obtained using SaPLIγand EcPLIγas parents.All these three types of cPLIγshowed 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 PLIγ.There was slight difference in the secondary structure of parental PLIγs and cPLIγs,For instance,C2（the isoforms with higher activity）and SaPLIγcompose of a six-amino acidα-helix from 150-155,while the length ofα-helix in other types of PLIγis less than 3 amino acids.PLIγfrom Bungarus multicinctus was found lack ofα-helix at the same position（150-155aa）,which has little anti-hemorrhagic activity.Therefore,α-helix at this specific position and its length might affect the activity of PLIγgreatly.Moreover,the number and position ofβ-sheet in PLIγmay also relate to the its activity in snake venom neutralization.However,these bio-informative analysis need further experimental validation.