PEGylation Of Recombinant Ganoderma Lucidum Immunomodulatory Protein(rLZ-8) And Purification Of Its Products

Recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8) is arecombinant protein expressed by Pichia pastoris eukaryotic expression system inour lab, possessing immunoregulation as well as anti-tumor effect, which have broadmarket prospects of State Category New Drug. Due to its small molecular weight,half-life of rLZ-8is very short in vivo. Furthermore, it has obvious immunogenicity.Pegylation can overcome these shortcomings, decreasing the immunogenicity,strengthening stability, and prolonging half-life features, thus improving theirpharmacokinetic properties, enhancing the pharmacodynamics and shortening thecycle of drug research period.In this study, the commonly used PEGylation technology was adopt and singlemethoxy polyethylene glycol propionic acid succinimidyl ester (mPEG-SPA5kDa)was selected as modifier to conjugate to lysine residues as well as the N-terminal ofrLZ-8to increase the molecular weight of rLZ-8, to prolong the serum half life of rLZ-8,and to enhance rLZ-8’s stability in vivo.For constructing detection method of PEG modification products, firstly, theproducts were separated by SDS-PAGE electrophoresis, then gels were bothCoomassie blue staining and Barium iodide staining. Coomassie brilliant blue onlystain protein but no PEG-specific staining, in contrast, Barium iodide only stain PEGbut no protein-specific staining of which brightness has a positive correlation withPEG amount. So, we can distinguish mPEG-SPA（no reaction）and modificatedmPEG-SPA(conjugated with rLZ-8). Two kind of stain done at the same time，theapproximate modification rate then can be calculate. High performance liquidchromatography analysis according to the molecular sieve principle plays a vital roleon the analysis of the modified product. Shimadzu (SHIMADZU) Shim-pack DIOL 150(25cm) chromatographic column was using and SuperdexTM75prep grade gelwas chosen as stationary phase. Samples were filtered through0.22μm filters,loaded20μl per gel, eluted by0.05M phosphate buffer (pH7.0), flowed by rate of1ml/min, and detected by280nm Wavelength. According to each component peaktime, peak height and peak area of the situation, modified point-digit of PEG could bepredicted approximately, which service for further research.In this study, physical and chemical properties of that rLZ-8and mPEG-SPA, thepH value of reaction system, molar ratio of that rLZ and mPEG-SPA, the ionicstrength of the reaction system, reaction temperature, reaction time and other factorswere investigate initially. Get the optimized modification reaction conditions of rLZ-8with mPEG-SPA by changing a single factor, controlling other factors constant,processing multi-level repeated trials. Then, Denaturing polyacrylamide gelelectrophoresis (SDS-PAGE) and high performance liquid chromatography (HPLC)analysis were utilized on the reaction products coming from various reactionconditions to determine the optimal conditions of single modified products. Theresults showed that modification reaction between mPEG-SPA and rLZ-8does notoccur under acidic conditions. And the modification reaction is easily in neutral andalkaline conditions, furthermore, the reaction mixed solution consists of four kinds ofproduct composition under pH8.0conditions. The bigger molar ratio between rLZ-8with mPEG-SPA, the more single protein products obtained; the smaller molar ratiobetween rLZ-8with mPEG-SPA, the more complex and the bigger molecular weightproducts obtained. Therefore, in order to get the smaller molecular weight of theuniform modified product, mole ratio between the two raw material was choose on1:1.As to the reaction time and temperature, due to the reaction is very easily,0.5h atroom temperature is enough to reach reaction balance, reaction product compositionno longer changes. Also，the ionic strength of the reaction product has little impact onproduct composition. In short, pH is the crucial factors that determine themodification reaction whether occur, and on pH8.0conditions, the molar ratio of tworaw materials is the important factor to determine the modification reaction productcomposition. The optimized reaction conditions:0.1M pH8.0sodium phosphatebuffer solution,1mg/ml protein final concentration,2h of reaction time.During the purification of modified products, SuperdexTM75prep grade gelfiltration chromatography were utilized; the mobile phase was0.05M phosphate- 0.15M of NaCl (pH7.0); flow rate was1ml/min; the detection wavelength was280nm,254nm,215nm The modify products were eluted with1.5column volumes onisoconcentration, and collected at a fixed volume (3ml) and peak collection (2ml),then the high purity modified products could be collected according to peak time ofeach component from high performance liquid chromatography (HPLC). Themodified rLZ-8may obtain up to98%purity using our purification method. Biologicalactivity of the product (rLZ-8) before and after modification was investigate by HL60cells agglutination test; T lymphocyte proliferation of the product (rLZ-8) before andafter modification was investigate by the BrdU assay. The results showed that thebiological activity of the modified products (mPEG-SPA modified rLZ-8) can beretained about70%of the original protein activity. As to the Characteristics of themodified products, the molecular weight was identified by Matrix-assisted laserdesorption/ionization time-of-flight spectrometry, modification sites of mPEG-SPA aswell. The results showed that the molecular weight of the modified product was17.9kDa, and the modification site of single-chain PEG-modified rLZ-8PEG isN-terminal. The experiments provide a scientific basis for pharmacodynamics andpharmacokinetics of rLZ-8and its modified products.In summary, the process in the issue is stable with good reproducibility andsuitable for large-scale production.