ZHANG Ji, WU Zhiyong, ZHU Lingyu, XIN Yu, GU Zhenghua, SHI Guiyang, ZHANG Liang
Human endostatin (hEDN), a bioactive polypeptide composed of 184 amino acids, is located in C-terminal of human collagen type XVⅢ. The molecular weight of hEDN is about 22 kDa. The three histidine residues at the N-terminal of hEDN and aspartic acid at position of 76 are four Zn2+ binding sites, which can bind to Zn2+ to play an important role in its anti-angiogenic activity. At present, the clinical application of hEDN is still facing challenges such as low solubility, poor stability, high price, and large doses. Therefore, exploring the efficient methods for the preparation of hEDN is of great significance because it is beneficial to expand its application in the field of medicine. With the development of genetic engineering technology, biological expression systems have been widely used in the expression and preparation of recombinant proteins or polypeptides. Recently, a variety of expression systems were developed to the expression of hEDN, such as mammalian cells, Pichia pastoris and Escherichia coli. The E. coli expression system has a lot of advantages, such as fast growth, clear genetic background, high expression level and low cost. For these reasons, E. coli expression system is one of the more suitable systems for the production of recombinant protein or polypeptide. However, the expression product of the gene hedn in E. coli is mostly insoluble inclusion bodies, which was formed due to protein misfolding. At the same time, the processing steps of inclusion bodies are usually complex and it is hard to refold the inclusion bodies successfully. Therefore, some fusion tags were selected to promote the soluble expression of hEDN in E. coli, such as GST tag, Trx A tag and MBP tag. This study aims to investigate the conditions of soluble expression of hEDN in E. coli. Furthermore, we tried to complete the large-scale preparation of hEDN using E. coli as a workhorse. The various expression systems including single gene, multiple genes, and fusion tags were constructed in E. coli BL21 (DE3) by using multiple molecular biological techniques, respectively. To verify the expression of hEDN, the recombinant strains harboring different expression systems and the controls were cultivated with a condition of 16 ℃,200 r/min, and then final concentration of 0.2 mmol/L IPTG was added to culture for inducing the expression of hEDN or fusion protein, respectively. After induction, the cell pellet was harvested and was lysed by ultrasonication. In order to investigate the expression of hEDN, the supernatant and insoluble substance were preliminary analyzed by using SDS-PAGE. Furthermore, MALDI-TOF/TOF was used to verify the expression of hEDN. The results showed that the recombinant strain BL21 (DE3)/pGEX-6p-1-hedn had successfully realized the soluble expression of fusion protein, which was consist of hEDN and the GST tag. Furthermore, the fermentation optimization was carried out to obtain high level expression of the hEDN fusion protein. Before the optimization, the optical density of the recombinant strain BL21(DE3)/pGEX-6p-1-hedn at 600 nm (OD600) was measured at the condition of 37 ℃, 200 r/min, and then the growth curve of was made. According to the growth curve, the expression of hEDN fusion protein was optimized by changing the inducing temperature, inducing time, IPTG dosage, and inducing point. Finally, the results showed that the optimum inducing conditions: 0.3 mmol/L IPTG was added into fermentation broth after cultivated 8-12 h and maintain the inducing temperature at 20 ℃ for 36 h. To obtain the fusion protein, the affinity chromatography system equipped with a PrePack GSH Purose 4 Fast Flow column was used to purify the hEDN fusion protein. Our results showed that the hEDN fusion protein with a GST tag can specifically bind to GST purification column and can be eluted. The eluent was further addressed by ultrafiltration for the replacement of buffer as well as concentration. The preliminarily purified sample was a fusion protein containing hEDN and GST tag, and the GST tag needs to be removed to obtain hEDN. Recombinant PreScission protease (PPase) was a fusion protein composed of human rhinovirus type 14 3C protease and GST tag. Definition of PPase activity (U): the amount of enzyme required to cleave 10 μg of GST-labeled fusion protein up to 90% or more at 5 ℃ for more than 16 h was defined as an activity unit. The protease can specifically recognize the short peptide Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro at low temperature, and it carries out its function between Gln and Gly amino acid residues. The hEDN fusion protein was verified by using the PPase. In theory, there are three main proteins could be obtained after enzymatic digestion, including PPase, GST tag and hEDN. According to the SDS-PAGE results, a target band about 22 kDa was obtained after PPase digestion. Furthermore, the target band was further confirmed as hEDN by MALDI-TOF/TOF analysis. This study provided an insight into the soluble expression and purification of polypeptides from human. Meanwhile, our work also provided a reference for the production of functional polypeptides. In the next step, the function of hEDN will be verified through cell biology experiments.
