Expression, Renaturation With Simultaneous Purification Of The Human Interferon-γ And Human Stem Cell Factor By Liquid Chromatography

Genetic engineering technology, especially the prokaryotic genetic engineering based on E. coli-expression systems, makes it possible manufacture almost any proteins with theoretical or applied significance in E. coli. However, in general, it is still a great challenge to obtain a purified protein product from E. coli, to satisfy clinical or research applications. One of the reasons is that, as the most important part of the "downstream technology", renaturation and purification of E. coli-expressed eukaryotic proteins are the "technological bottle-necks" of the prokaryotic engineering. It has accounted for about 70-90% of costs of a recombinant protein production. In this study, we firstly developed a high expression system and simplified technology for the production of recombinant human interferon-γ (rhIFN-γ) and recombinant human stem cell factor (rhSCF) in E. coli and then the obtained rhIFN-γ and rhSCF existing in inclusion body of the bacterial lysates were purified using liquid chromatography with simultaneous renatuation, respectively, resulting in rhIFN-γ and rhSCF with high purity and bioactivity.The thesis includes nine parts as follows. 1. ReviewE. coli expression system is the most widely used prokaryotic genetic engineering system due to its well defined genetic background, feasibility in manipulation, short amplification cycle, high production, as well as low cost. However, recombinant proteins expressed in E. coli exist mainly in the inclusion body. The renatuation as well as purification of recombinant proteins from E. coli lysates have been a "technological bottle-neck" for the production of recombinant pharmaceuticals in E. coli. The hSCF is a hematopoietic growth factor functioning in multiple hematopoietic lineages. When cooperating with other hematopoietic factors, hSCF shows a strong function of restoring hematopoiesis. High production and establishment of an efficient method for the renaturation and pufication of rhSCF are of significance not only for clinical utilization, but also for the production ofrecombinant proteins in E. coli.2. Refolding and high-level expression of human interferon-γ in E.coliMonocytes were obtained from health volunteers and total RNA was prepared from these cells after stimulation with LPS. The human interferon-γ (hIFN-γ) cDNA was amplified with RT-PCR and cloned into plasmid vector. DNA sequencing showed that the amplified hIFN-γ gene has two polimorphic sites. High-level expression of the rhIFN-y was achieved using temperature-controlled E. coli expression system. The expressed protein is about 55% of total cellular protein after induction. Pure rhIFN-γ protein was prepared with simultaneous refolding and purification using high performance hydrophobic chromatography (HPHIC) after fermentation of E. coli. The purified rhIFN-y has comparable bioactivity as its natural product.3. Cloning and expression of rhSCFThe hSCF cDNA was amplified by PCR using total cDNA from human lymph node as a template, and cloned. The 5'terminal of the hSCF cDNA was modified by degenerative PCR to obtain high-expression in E.coli. The hSCF cDNA was inserted into an E.coli expression vector pBV220, and was highly expressed in E. coli. The expressed rhSCF accounted for about 40% of total bacterial proteins under an optimal culture conditions.4. Fermentation of rhSCFAfter the high expression of rhSCF in E. coli, we established a fermentation protocol for a preparative-scale production of rhSCF. We optimized culture condition for the fermentation of the engineered E. coli expressing rhSCF, including the composition of the fermentation medium, culture temperature, stable growth condition, and the best feeding mode. The obtained results showed that, in a 5L-fermenter, under the optimized culture medium and step-feeding mode, the engineered bacterial strain produced highest amount of rhSCF and expressed in E. coli accounted for 40% of total bacterial proteins. After 11h of fermentation, the output of rhSCF was about 1.544gL-1. The study establishes a stable, high output, and short-period fermentation method for rhSCF.5. Dilution-renaturation and purification of rhSCF by weak anion exchangechromatography (WAX)The rhSCF has two intramolecular disulfide bonds between Cys4-Cys89 and Cys43-Cys138. E. coli-expressed rhSCF formed inclusion bodies, liking many other eukaryotic proteins. In this part of study, inclusion bodies of rhSCF were firstly renatured using a dilution method, and then purified by WAX. Inclusion bodies were recovered and solubilised in urea solution. We optimized the renaturation buffer, and under the optimized condition, the denatured rhSCF was efficiently renatured by dilution. The rhSCF refolded from an initial concentration of 3.65 g L-1 by the dilution gave a mass recovery of 38.1%, specific bioactivity of 4.27 ×105 IU mg-1, and purity of the refolded rhSCF was 85%. After purification using WAX, the purity of rhSCF was 95%, with a mass recovery of 76%, and specific bioactivity of 1.24× 106 IU mg-1.6. Resolution by liquid chromatographic column and cake used in renaturation with simultaneous purification of rhSCFTo renature and purify rhSCF using liquid chromatography, we firstly tested the resolution of chromatographic columns and cake with different lengths, using 6 kinds of standard proteins. The results showed that, the chromatographic cake in thickness 1 cm employed had almost the same resolution with usual chromatographic column of 10 cm in length. Although the obtained chromatographic peaks using chromatographic cake with large diameter and short length were not as sharp as those obtained from usual chromatographic column, the chromatographic cake could indeed satisfy with the request of the large-scale separation and purification of biological macro-molecules in biotechnology. We also renatured with simultaneously purified rhSCF using two types of HPHIC cakes or unit of simultaneous renaturation and purification of proteins (USRPP)7. Optimization of renaturation with simultaneous purification of rhSCF using chromatographic cakeA renaturation with simultaneous purification method of rhSCF expressed in E. coli. with HPHIC was investigated in this part. Different factors effecting on the renaturation and simultaneous purification, including the types of the stationary and mobile phases of HPHIC, components of the mobile phase, were investigated using ananalytical column. The optimal condition for the renaturation with simultaneous purification of rhSCF using the HPHIC cake was also studied/The obtained results showed by the chromatographic cake that, compared with the traditional dilution refolding method, by one step in 40 min, the purity of the renatured rhSCF was more than 94.5%, specific bioactivity of 1.28 × 10 6IUmg-1, and mass recovery of 36.5%. The total bioactivity recovery was 3-folds higher than that of the usual dilution method. These results provided further evidence that HPHIC is a useful tool in the refolding and purification of recombinant proteins.8. Renaturation with simultaneous purification of rhSCF using urea gradient SECWe studied the application of the urea gradient in SEC for the renaturation and purification of rhSCF, according to that protein and small molecules migrate differentially in gel filtration. The results showed that, the urea gradient in SEC improved the mass and bioactivity recovery of renatured rhSCF significantly. During the chromatographic process, a linear elimination of urea provided a better environment for renaturation of the denatured rhSCF. We also found that elution-time, flow-speed, and the rhSCF concentration are key factors influencing its renaturation9. Renaturation of rhSCF by the microcapisule methodBesides the fregoing liquid chromatographic method, we also investigated the renaturation by microcapisule method. Inclusion bodies were recovered and solubilized in urea solution. For a fast refolding rhSCF with low cost, up to 148.6 mg of denatured inclusion bodies were refolded by diluting to 20-fold, according to the microcapsule release renaturation protocol. The rhSCF refolded from an initial concentration of 3.65 g L-1 by the microcapsule dilution gave a mass recovery of more than 40%, with specific activity of 8.72×105IU.mg-1. The finally refolded rhSCF product was tested by reverse-phase liquid chromatography and its molecular weight was measured by MALDI-TOF-MS. The results indicated that in terms of the mass recovery and purity of the rhSCF, the microcapsule dilution method is a feasible method and better than the usual dilution method.In summary, in the presented studies, we have cloned and highly expressed rhSCF in E. coli and established a fermentation method to produce rhSCF in E. coli at a preparative scale. The renaturation with simultaneous purification of rhSCF using various chromatographic methods, even in preparative scale were investigated in details. These studies provided essential technologies for industrial production of rhSCF, and contributed to the improvement of downstream technology of genetic engineering.