1.Efficient fusion expression of G13 domain derived from granulysin in Escherichia coli.
Xiaoqiang LIU ; Xiangdong ZHA ; Yazhong XIAO ; Jinhuan YANG ; Nengshu LI
Chinese Journal of Biotechnology 2009;25(2):235-241
The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.
Anti-Infective Agents
;
metabolism
;
Antigens, Differentiation, T-Lymphocyte
;
genetics
;
Cyanogen Bromide
;
pharmacology
;
Escherichia coli
;
genetics
;
metabolism
;
GTP-Binding Protein alpha Subunits, G12-G13
;
biosynthesis
;
genetics
;
Inclusion Bodies
;
metabolism
;
Protein Structure, Tertiary
;
genetics
;
Recombinant Fusion Proteins
;
biosynthesis
;
genetics
;
Thioredoxins
;
genetics
;
Transfection
2.Galpha12 Protects Vascular Endothelial Cells from Serum Withdrawal-Induced Apoptosis through Regulation of miR-155.
Hyeon Jeong LEE ; Eun Jig LEE ; Miran SEO
Yonsei Medical Journal 2016;57(1):247-253
PURPOSE: Apoptosis of vascular endothelial cells is a type of endothelial damage that is associated with the pathogenesis of cardiovascular diseases such as atherosclerosis. Heterotrimeric GTP-binding proteins (G proteins), including the alpha 12 subunit of G protein (Galpha12), have been found to modulate cellular proliferation, differentiation, and apoptosis of numerous cell types. However, the role of Galpha12 in the regulation of apoptosis of vascular cells has not been elucidated. We investigated the role of Galpha12 in serum withdrawal-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and its underlying mechanisms. MATERIALS AND METHODS: HUVECs were transfected with Galpha12 small-interfering RNA (siRNA) to knockdown the endogenous Galpha12 expression and were serum-deprived for 6 h to induce apoptosis. The apoptosis of HUVECs were assessed by Western blotting and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expressions of microRNAs were analyzed by quantitative real-time PCR. RESULTS: Knockdown of Galpha12 with siRNA augmented the serum withdrawal-induced apoptosis of HUVECs and markedly repressed the expression of microRNA-155 (miR-155). Serum withdrawal-induced apoptosis of HUVECs was inhibited by the overexpression of miR-155 and increased significantly due to the inhibition of miR-155. Notably, the elevation of miR-155 expression prevented increased apoptosis of Galpha12-deficient HUVECs. CONCLUSION: From these results, we conclude that Galpha12 protects HUVECs from serum withdrawal-induced apoptosis by retaining miR-155 expression. This suggests that Galpha12 might play a protective role in vascular endothelial cells by regulating the expression of microRNAs.
*Apoptosis
;
Atherosclerosis/*blood/genetics/immunology
;
Cell Proliferation
;
Endothelial Cells/*metabolism
;
GTP-Binding Protein alpha Subunits, G12-G13/*genetics
;
Gene Expression Profiling
;
Gene Expression Regulation
;
Human Umbilical Vein Endothelial Cells/cytology
;
Humans
;
MicroRNAs/*metabolism
;
Protective Agents
;
*RNA, Small Interfering
;
Real-Time Polymerase Chain Reaction
;
*Transfection