Chemicals and strains and plasmids
Terrific Broth (TB) and kanamycin were purchased from ThermoFisher Scientific, Chengdu, China for culturing bacterial strains. PCR kit, restriction enzymes, and gel extraction kit were purchased from TaKaRa Co. Ltd., Beijing, China. The primary antibody, mouse anti-iLRP and the secondary antibody, goat anti-mouse IgG conjugated with Horseradish peroxidase (HRP) were bought from Abcam, Shanghai, China. Other molecular biology reagents were from Solarbio Science & Technology Co. Ltd., Beijing, China. All chemicals in this study were with analytical grade or a higher purity.
Escherichia coli DH5α was used as the host for recombinant DNA manipulation, and BL21(DE3) was used for gene expression. Competent cells of bacterial strains were purchased from ThermoFisher Scientific, Chengdu, China. Plasmids pUC57 and pET30a(+) for cloning and expression were from ChinaPeptides, Shanghai, China. The plasmid pENTER containing the natural human iLRP cDNA was bought from Vigene, Shandong, China. All hosts and plasmids were stored at -80 °C in our lab.
The technology of codon optimization has been a valuable tool for producing proteins as therapeutic agents or research reagents in heterologous host. With the availability of large amount of genomics data, and with the increased knowledge of protein expression, function and structure relationships, gene expression levels can be significantly improved by this technique [8, 21]. During the process of applying the technique, a variety of key elements involved in different stages of protein expression, like as GC content, codon adaptability, mRNA structure, and various cis-elements in transcription and translation should be carefully taken into consideration [9, 22]. In this study, human iLRP gene was optimized in terms of above points using a commercial proprietary algorithm, NG® Codon Optimization Technology (Synbio Technologies, NJ, USA) in order to analyze its expression in Escherichia coli.
Construction of expression vectors
Plasmid pET-30a(+) (from Novagen) was used as the backbone for the construction of vectors in this study. The vector pET-His-iLRP was constructed by inserting the natural iLRP gene between restriction sites of NdeI and XhoI. The natural iLRP gene was amplified by PCR using the plasmid pENTER (Vigene, Shandong, China) containing the human iLRP gene  as template. In order to add a restriction site of NdeI and a 6xHis-Tag to the N-terminal of iLRP, the 5'-primer sequence was synthesized by Sangon Biotech, Shanghai, China for this PCR with the sequence as: 5'-ATACATATGCACCATCATCATCATCATTCCGGAGCCCTTGATG- 3'. Similarly, a restriction site of XhoI was added to the 3'-primer, and the sequence is: 5'-CCGCTCGAGTTAAGACCAGTCAGTGGTTGC-3'. To construct the vector pET-His-S-iLRP, the restriction sites of BglIIand XhoI were used for the process of cloning so that the original 6xHis-Tag and the S-Tag were kept as it was. Therefore, the 5'-primer for amplifying the iLRP gene from pENTER was designed as: 5'-ACGAGATCTTCCGGAGCCCTTGATG-3', and the 3'-primer is same as above. PCR was both performed at the following conditions: initial denaturation at 98°C for 2 min, 30 cycles of denaturation at 98°C for 40 s, annealing at 58°C for 40 s, and extension at 72°C for 1 min, and the final extension at 72°C for 4 min and then maintenance at 4°C forever. In the construction of pET-His-Opt-iLRP, the codon-optimized iLRP gene with the additional elements of restriction site of NdeI and the 6xHis-Tag at 5'-end of iLRP and EcoRV at the 3'-end of iLRP was synthesized by ChinaPeptides, Shanghai, China and then cloned into pET-30a(+) between NdeI and EcoRV. All constructed vectors were characterized by sequencing and digestion of restriction enzymes.
Above vectors were transformed into Escherichia coli BL21 (DE3) (from Novagen) for the analysis of protein expression at a small scale. Briefly, on day 1, a fresh TB agar plate containing 50 μg/ml kanamycin was inoculated with bacterial stocks for each vector. On day 2, well isolated single clones were picked from the plates and cultured overnight in test tubes with 3 ml TB containing 50 μg/ml kanamycin, shaking at the speed of 200 rpm and at 37°C. In the morning of day 3, overnight cultures were transferred to 500 ml flasks with 100 ml fresh TB and 50 μg/ml kanamycin, continuously shaking at the speed of 200 rpm and at 37°C until the optical density at 600 nm (OD600) reached to 1.0. At this point, a small aliquot of cultures for each sample was collected as negative control. Then, the rest of cultures was induced by adding isopropyl-thio-β-D-galactoside (IPTG) to a final concentration of 1.0 mM and shaking at the conditions above until the OD600 reached to the same point of 6.0. Cell cultures were harvested by centrifuging at 4°C, 5000 rpm for 10 min and stored at -80°C.
SDS-PAGE and Western Blotting
Cell pellets harvested above were divided into two parts. One of them was directly lysed to prepare samples of whole cell lysates for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and another one was used to prepare inclusion bodies. Cell pellets were washed with ice-cold phosphate-buffered saline (PBS, PH 7.2) twice, and resuspended with 5 ml of PBS. The cells in suspension were disrupted by sonication with 20 s pulse-on and pulse-off time each using an ultrasonicator (Ningbo Scientz Biotechnology Co. LTD, China). The inclusion bodies were then isolated by centrifugation at 12,000 rpm for 20 min at 4°C. After washing twice with 20 ml ice-cold PBS, the inclusion bodies were dissolved in 5 ml of solubilization buffer (10 mM Tris-HCl, 100 mM sodilum phosphate, 6 M urea, 2 mM 2-mercaptoethanol, PH 8.0). After centrifugation at 12,000 rpm for 20 min, the total protein in the supernatant was determined with a BCA protein assay kit (ThermoFisher Scientific, Chengdu, China), and then, the supernatant was used to prepare the samples for SDS-PAGE with equal protein amount of each sample being loaded onto the gel. All samples for SDS-PAGE were boiled in 1x reducing loading buffer at 98°C for 10 min and subjected to 12% denaturing SDS-PAGE. Gels were stained by Coomassie Brilliant R-250 (ThermoFisher Scientific, Chengdu, China) to identify the expected protein bands. For Western Blotting, gels were blotted to nitrocellulose membrane, and then the membrane was blocked with 2.5% (w/v) skimmed milk powder in PBS, pH 7.2 for 2 h at room temperature (RT), and successively incubated with mouse anti-iLRP monoclonal antibody (Abcam, Shanghai, China) at a dilution of 1:5,000 at 4°C overnight. Next day, the membrane was washed 3 times, and then incubated with a 1:10,000 dilution of horseradish peroxidase (HRP) conjugated anti-mouse IgG secondary antibody at RT for 2 h. After the incubation, wash the membrane 5 times, then incubate the membrane with electrochemiluminescence (ECL) reagent (ThermoFisher Scientific, Chengdu, China) for 5 min in dark. Protein bands were detected by ChemiDocTM MP imaging System (Bio-Rad Laboratories Co. LTD, Shanghai, China).