Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification

Abstract

Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.

Figure 1. Construction and schematic representation of the His8-hLIF, PDI-hLIF, and PDIb'a'-hLIF expression vectors.

(A) Vector maps of His8-hLIF, PDI-hLIF, and PDIb'a'-hLIF. Fusion protein expression in E. coli is controlled by the IPTG-inducible T7 promoter, and the selection markers for His8-hLIF for PDI-hLIF are kanamycin and ampicillin, respectively. (B) Schematic structure of the His8-hLIF, PDI-hLIF, and PDIb'a'-hLIF fusion proteins. The arrows indicate the recognition sites for the proteolytic cleavage of TEV protease. The black bars indicate the additional sequences that resulted from Gateway plasmid recombination. The lengths of each domain are scaled to the length ratio of each domain.

Figure 2. SDS-PAGE analysis of hLIF fused with three different tags in E. coli.

Protein expression was induced by 0.5 mM IPTG at (A) 37°C and (B) 18°C. Arrows indicate the hLIF proteins fused with each tag. Equal amounts of cells were loaded into the C and I lanes. For the P and S lanes, the volume of the precipitant obtained after sonication and centrifugation was adjusted to ensure that it was obtained from the same amount of cells. 10 μl of sample was loaded. M, molecular weight size marker; C, total cellular protein before IPTG induction (control); I, total cellular protein after IPTG induction; P, insoluble cell pellet; S, soluble supernatant after sonication.

Figure 3. Purification of hLIF from E. coli.

Purified (A) His8-LIF and (C) PDIb'a'-hLIF obtained from E. coli were analyzed using SDS-PAGE. Lanes 5 and 5' show the cleavage of His8 and PDIb'a'. M, molecular weight marker; lanes1 and 1', total cells before IPTG induction (negative control); lanes 2 and 2', total cells treated with IPTG; lanes 3 and 3', soluble fraction after sonication; lane 4, His8-hLIF fusion protein purified using HisTrap HP(25 kDa); lane 4', PDIb'a'-hLIF fusion protein purified using ion-exchange chromatography (55.4 kDa); lane 5, His8 tag cleaved using TEV protease; lane 5', PDIb'a' tag cleaved using TEV protease; lanes 6 and 6', final purified hLIF. The amount of loaded protein was individually adjusted for each lane in order to make the bands clearly visible. (B, E) Silver-stained gel from SDS-PAGE used to assess purified hLIF: 1.6 μg and 1.4 μg hLIF obtained from His8-hLIF and PDIb'a'-hLIF were loaded into lanes 7 and 7', respectively. (D) Gel filtration chromatogram of PDIb'a'-hLIF after cleavage. hLIF and PDIb'a' were separated according to size. The TEV protease eluted with hLIF.

Figure 4. Mass analysis of hLIF purified from E. coli.

(A) Tryptic peptide map of hLIF (181 aa). Reduced (B) and nonreduced (C) hLIF proteins cleaved from PDIb'a'-hLIF were analyzed using MALDI-TOF MS. Peptide fragments digested by trypsin are shown as red dotted lines on the schematic representation inset.

Figure 5. Biological activity.

(A) ESCs demonstrated equivalent growth following treatment with control LIF, hLIF from His8-hLIF, and hLIF from hPDIb'a'-hLIF (upper). ESCs demonstrated SSEA-1 expression on the cell surface in the presence of LIFs (middle). The expression levels of two stem cell factors, nanog and Oct3/4, in ESCs treated with LIFs were quantified using qRT-PCR (lower). (B) His8-hLIF-treated cells, like those treated with control LIF, maintained an ESC-like appearance (left and middle). SSEA-1 expression in response to noncleaved His8-hLIF was similar to control LIF (right). (C) Schematic diagram describing ESC maintenance and differentiation processes (upper). Cellular morphology was effected by the withdrawal of LIF (upper right) compared with Figures 5A and 5C. The removal of LIFs induced two differentiation markers, nestin and brachyury, which were expressed at similar or higher levels (middle). ESCs gave rise to Flk-1⁺ mesodermal precursor cells following His8-hLIF treatment (lower).