Construction, Screening And Structure Optimization Of Recombinant STNFα - RI Protein Synonymous Password Mutant

Tumor necrosis factor-TNF-, chiefly produced by activated macrophages, is amajor imflammatory cytokine that contributes to rheumatoid arthritis. Soluble tumornecrosis factor-sTNF is generated via proteolytic cleavage of the membrane-integratedTNF-by metalloprotease TNF-converting enzyme. It exists as a bioactive homotrimerin vivo.TNF-can bind to two receptors(TNF-RI and TNF-RII)on synovial cells,macrophages, chondrocytes and osteoclasts. When activated, these cells produce matrixmetalloproteinases, collagenase and stromelysin, resulting in local inflammation thatfurther leads to cartilage destruction and bone erosion. Over the recent ten years, there hasbeen a rapid development of biological drugs that target sTNF Among the world’s top tenbest selling drugs in2013, three are anti-sTNF macromolecular drugs intended fortreatment of rheumatoid arthritis(Humira,Remicade,Enbrel). Humira,Remicade areanti-TNF antibodies, while Enbrel is an artificially engineered dimeric fusion proteindeveloped from TNF-RII. While these drugs have proven to be effective in clinicaltreatment of rheumatoid arthritis, studies have shown that long-term administration cancause infections and tumors. Furthermore, mammalian cell culture productions of thesedrugs generally have high manufacturing cost and low productivity, in addition to thedifficulties associated with quality control of glycosylation modifications of proteins. Thesale price of such imported drugs is averaged to around20,000RMB per dose, Which ishardly affordable for the mass majority of patients in China. Therefore, the exploitation ofnew efficient and safe rheumatoid arthritis drugs becomes a major focus of currentresearch.This study is based on the development of recombinant protein sTNF-RI(Sop55-2.6). The aim of this study is to improve the soluble expression of sTNF-RI(Sop55-2.6) as well as its yield in inclusion body, through design and optimization of itsmolecular structure in genetic and protein aspects. The main content of this paper included:(1) Improvement of yield of sTNF-RI (Sop55-2.6) in bacteria inclusion body byoptimization of the DNA coding sequence of the target protein using a novel technology of synonymous codon mutation library construction and screening.(2) Improvement ofsoluble expression of target protein in bacteria host through silico analysis of thestructure-function relationship of target protein, and design and optimize the recombinantprotein sTNF-RI in the following aspects: molecular truncation, expression vectors, fusiontags, amino acid mutations and expression host.A synonymous-codon mutation library of sTNF-RI was first constructed using therecombinant tumor factor-receptor I gene as a template. Degenerate primers weredesigned and PCR conditions were optimized to generate a mutant library of1013in size.Screening of445randomly picked clones from the library yielded45unique clones withhigher target protein yield in inclusion bodies compared to that of the wild type.Sequencing showed that42out of the45unique clones had the same amino acid sequencewith the wild type. Target protein yield in the top mutant clone obtained was2.44timeshigher than that of the wild type clone.To increase soluble expression of target protein in bacteria, Sop55-2.0mutant proteinwas designed by removing the19amino acids at the C-terminal of wild type protein. DNACoding sequence of the mutant protein was then cloned into pET22b (+) vector for solubleexpression in periplasm. Preliminary ELISA analysis of binding affinity revealed an EC50value of337.4g/ml. Next, soluble expression of Sop55-2.0protein was achieved by fusionto Trx-tag using pET32a (+) vector. Crude Sop55-2.0from supernatant was purified andTrx-tag removed by Enterokinase digestion. The EC50of the purified protein (purity>85%)was determined to be about154.6g/ml using ELISA. To further improve the affinity andsolubility of truncated protein,17amino acids at the N-terminal on Sop55-2.0was removedto obtain Sop55-2domain protein. Sop55-2.0, Sop55-2domain and Sop55-2.6achievedsoluble expression with Fc binding diploid in CHO cells. The purity of the proteins wereabove95%. L929cytology detection showed that the binding affinity of Sop55-2.6-Fc wasclose to that of Sop55-2.6; However, both Sop55-2domain-Fc and Sop55-2.0-Fc showedno significant binding activity. We concluded that Sop55-2.6was a potential candidate forfurther development.To further improve soluble expression of Sop55-2.6in cytoplasm, fusion of targetprotein to3×Flag tag and multi-point mutations of the amino acid sequence were performed. Yield of target protein by soluble expression was estimated to be20%of thetotal protein in supernatant. The EC50of the optimized fusion protein was about4.4g/mlmeasured by ELISA. The cytologic efficacy of the soluble protein has yet to be determined.In this study, a novel technology of synonymous codon library construction andscreening was adopted to increase the yield of the target protein by replacing originalcodons in the wild type protein with optimized synonymous codons. In addition, in silicostructural analysis facilitated the design of truncated protein, selection of expression vectorand fusion tag, and optimization of amino acid sequence and host strain. Finally, Sop55-2.6obtained in our study served as a potential candidate for further drug development. Thesoluble expression of this protein was achieved in E.coli. This study provided valuablereference for the structural design and optimization of recombinant protein.