Abstract
Human GM-CSF is a cytokine consisting of 127 amino acid residues, with four cysteines being involved in two disulfide bonds. Although GM-CSF is glycosylated in its natural form, the glycosylation perhaps has not been involved in its biological function. GM-CSF stimulates the survival, proliferation, and differentiation of hem at opoietic progenitor cells and also enhances the functional properties of mature myeloid cells. GM-CSF is used as a therapeutic agent in various clinical cases such as neutropenia following chemotherapy, bone marrow transplantation, acute myeloid leukemia… In this study, we report the results on the cloning and expression of recombinant human GM-CSF in the periplasmic space of Escherichia coli. The hGM-CSF gene was amplified by polymerase chain reaction using two oligonucleotide primers containing NcoI and XhoI restriction sites. This DNA fragment was successfully cloned between the NcoI and Xho I sites of the plasmid pET-22b, in frame with the pelB signal peptide sequence. The expression vector pET-hGM was transformed into E. coli BL21(DE3) and the transformants were induced by IPTG and examined for hGM-CSF production. Periplasmic proteins were released by osmotic shock treatment. The expression of recombinant hGM-CSF was evaluated by SDS–PAGE in total, cytoplasmic and periplasmic fractions. The recombinant hGMCSF in periplasmic fraction was then subjected to ion exchange chromatography using Q Sepharose FF column with saltincrement elution step. SDS-PAGE showed there was a visible expression of recombinant hGM-CSF in the periplasmic fraction of the E. coli BL21(DE3)/pET-hGM and a purified band with the purity of 97.4% after ion exchange chromatography. This result was further confirmed by Western blot using anti-hGM-CSF antibody.
Highlights
Its natural form, the glycosylation perhaps has not been involved in its biological function
Human GM-CSF is a cytokine consisting of 127 amino acid residues, with four cysteines being involved in two disulfide bonds
GM-CSF is glycosylated in its natural form, the glycosylation perhaps has not been involved in its biological function
Summary
HsdR17 (rk-/mk-) supE44 thi - recA1 gyrA96 lacU169 ( 80 lacZ M15)] (Takara) được sử dụng để tạo dòng và lưu trữ plasmid. Sản phẩm PCR được xử lý tạo đầu dính với hai enzyme cắt giới hạn trên và tinh chế bằng EZ-10 Spin Column DNA Gel. Extraction kit (BioBasic, Canada) theo hướng dẫn của nhà sản xuất. Kích thước của đoạn DNA mang gen hgm-csf và plasmid pET-22b đã cắt mở vòng được xác định bằng điện di trên gel agarose 1 %. Sàng lọc thể biến nạp và kiểm tra bằng enzyme cắt giới hạn và PCR plasmid Các thể biến nạp DH5α mọc được trên môi trường LBA-Amp tiếp tục được sàng lọc sơ bộ bằng phương pháp PCR khuẩn lạc với cặp mồi 5’-Nco và 3’-Xho. Thu nhận thể biến nạp cho kết quả PCR dương tính và tách chiết plasmid. Phân tích kết quả bằng điện di SDS-PAGE và lai với kháng thể đặc hiệu kháng hGM-CSF
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