Process Optimization of PEGylating Fused Protein of LL-37 and Interferon-α2a_Journal of Biomedical Engineering

Authors：

 ZHANGMingjie

Pharmaceutical Co., Ltd. Guangdong Zijing Zhengtian, Heyuan 517000, China;

Corresponding author：

ZHANGMingjie, Email: mingjiezh@126.com

Keywords：

interferon-α2a; LL-37; polyethylene glycol (PEG); PEGylating

DOI：

10.7507/1001-5515.20150224

Video：

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PEGylating is an effective way for prolonging the half-time period and decreasing the immunogenicity of protein drugs. With experiments of single factor, it was proved that the optimal processes for PEGylating the fused protein of LL-37 and interferon (IFN)-α2a were:PEG molecular weight was 5 000, fused protein concentration was 0.6 mg/mL, the mole ratio of protein to mPEG₅₀₀₀-SS was 1:10, the reaction temperature was 4℃, and the pH was 9.0, respectively. With orthogonal experiments, we proved that the influential order of 3 main factors is:the fused protein concentration > the mole ratio of protein and mPEG₅₀₀₀-SS > pH and the optimal conditions were the fused protein concentration as 0.6 mg/mL, the mole ratio of protein and mPEG₅₀₀₀-SS as 1:10, pH as 8.8. Under these optimal conditions, the average rate of PEGylated protein with 3 times parallel experiments was 86.98%. After PEGylated, the interferon activity and antimicrobial activity of fused protein could be remained higher than 58% and 97%, respectively.

Citation： ZHANGMingjie. Process Optimization of PEGylating Fused Protein of LL-37 and Interferon-α2a. Journal of Biomedical Engineering, 2015, 32(6): 1261-1266. doi: 10.7507/1001-5515.20150224 Copy

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1. SHINNAR A E, BUTLER K L, PARK H J. Cathelicidin family of antimicrobial peptides:proteolytic processing and protease resistance[J]. Bioorganic chemistry, 2003, 31(6):425-436.
2. STEINSTRAESSER L, KOEHLER T, JACOBSEN F, et al. Host defense peptides in wound healing[J]. Molecular Medicine, 2008, 14(7-8):528-537.
3. MOLHOEK E M, DEN HERTOG A L, DE VRIES A M, et al. Structure-function relationship of the human antimicrobial peptide LL-37 and LL-37 fragments in the modulation of TLR responses[J]. Biol Chem, 2009, 390(4):295-303.
4. HENZLER WILDMAN K A, LEE D K, RAMAMOORTHY A. Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37[J]. Biochemistry, 2003, 42(21):6545-6558.
5. JONASCH E, HALUSKA F G. Interferon in oncological practice:review of interferon biology, clinical applications, and toxicities[J]. Oncologist, 2001, 6(1):34-55.
6. CUMMINS J M, KRAKOWKA G S, THOMPSON C G. Systemic effects of interferons after oral administration in animals and humans[J]. Am J Vet Res, 2005, 66(1):164-176.
7. OSBORN B L, OLSEN H S, NARDELLI B, et al. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-αfusion protein in cynomolgus monkeys[J]. J Pharmacol Exp Ther, 2002, 303(2):540-548.
8. RABHI-ESSAFI I, SADOK A, KHALAF N A. A strategy for high-level expression of soluble and functional human interferonαas a GST-fusion protein in E.coli[J]. Protein Engineering Design & Selection, 2007, 20(5):201-209.
9. WANG Yu-sen, YOUNGSTER S, GRACE M, et al. Structural and biological characterization of pegylated recombinant interferonα-2b and its therapeutic implications[J]. Adv Drug Deliv Rev, 2002, 54(4):547-570.
10. KURFURST M M. Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after Sodium dodecyl sulfate-polyacry-lamide gel electrophoresis[J]. Anal Biochem, 1992, 200(2):244-248.
11. 杨云霞, 朱玲, 王伯瑶, 等.人抗菌肽FALL-39突变分子的特性和抗菌功能的比较[J].华西药学杂志, 2006, 21(5):422-424.
12. LEE B K, KWON J S, KIM H J, et al. Solid-phase PEGylation of recombinant interferonα-2a for site-specific modification:process performance, characterization, and in vitro bioactivity[J]. Bioconjug Chem, 2007, 18(6):1728-1734.
13. ODAIMI M, JASSOUS I EI, RICHA A, et al. PhaseⅠ/Ⅱtrial of a novel regimem of GSM-CSF, IL-2 and pegylated interferon-alpha 2b (PEG-intron) for stageⅣmelanoma and renal cell carcinoma (RCC)[J]. Journal of Clinical Oncology, 2004, 22(14):7573-7579.
14. MAISER B, DISMER F, HUBBUCH J. Optimization of random PEGylation reactions by means of high throughput screening[J]. Biotechnol Bioeng, 2014, 111(1):104-114.

Journal of Biomedical Engineering

Process Optimization of PEGylating Fused Protein of LL-37 and Interferon-α2a

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