Reference Standard Preparation For The Recombinant Ganoderma Lucidum Immunomodulatory Protein (rlz-8)

The Ganoderma lucidum immunomodulatory protein Ling Zhi-8 (LZ-8) extracted from Ganoderma lucidum mycelium is first found and named by Kino's team from Japan and is the first known member of the Fungal Immunomodulatory Protein (FIP) family. The Task Force has re-designed the coding genes of LZ-8 based on the bias of yeast genetic code to obtain the efficiently expressed recombinant Ganoderma Lucidum immunomodulatory protein (rLZ-8). The study is dedicated to establish the preparation and quality control methods for rLZ-8.1. Reference standard preparation for rLZ-8The preparation procedures include five main steps, namely, preparation of protein material, protein capture, moderate protein purification, polishing protein purification and freeze-drying. The main principle of purification process is to minimize experimental procedures based on the premise of the efficiency of protein purification to minimize the possibility of activity and quantity loss of the protein.①Preparation of protein material:First, to remove the yeast mycelium and some pigment by microfiltration;Then,to remove most of the remaining pigments and salts and eventually condensing the broth to 6L by ultrafiltration. This stage allows the content of rLZ-8 protein in the sample up to 15%.②Protein capture:using cationic exchange chromatography (GE, XK 26/20 type, SP Sapharose XL ) with pH 3.5 50 mM NaAc-HAc buffer solution as A phase and pH 3.5 50 mM NaAc-HAc/1M NaCl buffer solution as B phase, the flow rate is 5 ml/min, linear gradient elution, the sample volume is 200ml. This stage can remove most unwanted proteins, small molecules nucleic acid segments and pigment. The rLZ-8 protein content in the sample can reach 45%.③Moderate protein purification: using a strong anion-exchange chromatography (Q-Sapharose Fast Flow) with pH 7.2 in 25 mM Tris-HCl buffer as A phase and pH 7.2 in 25 mM Tris-HCl 0.5 M NaCl buffer as B phase,the flow rate is 3 ml/min, the sample volume is 5 ml. This stage can further remove unwanted proteins and concentrate the target protein effectively. The rLZ-8 protein content is≥99%.④Polishing protein purification:using a gel chromatography (SuperdexTM 75) with mobile phase of pH 7.2 50 mM Tris-HCl/0.15M NaCl, the flow rate is 3 ml/min, the sample volume is 5 ml. Pure rLZ-8 protein can be obtained in this phase, namely the protein purity is≥99.99%.⑤freeze-drying stage:bottle-packing 0.5ml samples each in 2ml vials to obtain 1 mg/bottle rLZ-8 protein standard by freeze-drying.2. Quality control of rLZ-8 standard sample The quality control of rLZ-8 standard sample involves moisture determination, immunological detection, Western blot, protein purity testing, protein content testing, peptide mapping, isoelectric point testing, N-terminal amino acid sequencing and molecular weight identification using laser desorption time of flight mass spectrometry.①moisture determination:As the standard goes, the protein standard moisture content of freeze-dried formulations should be less than 1%. The moisture content in this experiment is 0.91%, in line with standards.②immunological detection: Results show that, rLZ-8 can trigger sheep red blood cell aggregation in a certain range of concentration while have no effect on the four blood types of human blood cells. When added in 1% sheep red blood cells, rLZ-8 begins to induce coagulation at a concentration of 1.5μg/ml and induces complete agglutination of sheep red blood cells at a concentration of 4.6μg/ml.③UV absorption experiment:rLZ-8 protein has the strongest absorption peak in the 280nm±2nm, in line with standards.④Purity Determination Experiment:Reference standards should be free of unwanted proteins and other impurities besides added man-made materials. The rLZ-8 protein purity reaches 100% using HPLC detection.⑤protein determination:Standard curve was determined produced by Lowry's according to the 2010 Chinese Pharmacopoeia III, the same as the rLZ-8 protein content. The result showed that the protein content of the rLZ-8 standard prepared in this experiment is 1.0091μg/ml⑥Isoelectric point determination:The experimental calculated isoelectric point of rLZ-8 standard is 4.68.⑦N-terminal amino acid sequence analysis:The N-terminal sequence of rLZ-8 standard prepared in this experiment is SDTALIFRLAWD, which is consistent with the theoretical value.⑧Molecular weight determination:The molecular weight of rLZ-8 identificated by laser desorption time of flight mass spectrometry is 12510.7Da, in line with the expected results.The experiment has established the preparation, quality assessment and control methods of rLZ-8 standard sample. These studies can be used for drug R & D process identification and purity/activity researching of the pharmaceutical raw materials and finished products, to provide reference for the quality control of its APIs and finished drug in follow-up study.