Integrated refolding techniques for Schistosoma japonicum MTH1 overexpressed as inclusion bodies in Escherichia coli.

The full-length cDNA of MTH1in Schistosoma japonicum was previously isolated. However, insoluble protein expression in Escherichia coli is the biggest bottleneck limiting biological and biophysical studies. Protein aggregation could not be significantly prevented using solubilization or refolding techniques, and denatured MTH1 protein could not be refolded to the native monomer form. Hence, integrating several refolding techniques within the protein refolding process of MTH1, a large amount of active MTH1 was obtained for protein crystallization. We primarily utilized the two-step-denaturing and refolding method and the protein refolding screening technique, as well as the continuous dialysis method. First, we identified the refolding buffer composition that allowed for successful refolding to overcome protein precipitation. Next, we used the two-step-denaturing and refolding method and the continuous dialysis method to suppress protein aggregation. In the end, we obtained 15 mg of active MTH1 monomer with 95% purity from 0.5l medium. Integrated refolding techniques proved to be excellent for obtaining the native monomer of S. japonicum MTH1 from inclusion bodies, paving the way for future biological and biophysical studies.

Identification and expression analysis of genes in response to high-salinity and drought stresses in Brassica napus.

High salinity and drought are the major abiotic stresses that adversely affect plant growth and agricultural productivity. To investigate genes that are involved in response to abiotic stresses in Brassica napus, a comprehensive survey of genes induced by high-salinity and drought stresses was done by macroarray analysis. In total, 536 clones were identified to be putative high-salinity-or drought-responsive genes. Among them, 172 and 288 clones are detected to be putative high-salinity- and drought-inducible genes, whereas 141 and 189 are candidates for high-salinity- and drought-suppressed genes, respectively. The functional classification of these genes are indicated that belonged to gene families encoding metabolic enzymes, regulatory factors, components of signal transduction, hormone responses, some abiotic stresses-related proteins, and other processes related to growth and development of B. napus. From the upregulated candidate genes, some interested genes were further demonstrated to be high-salinity- or/and drought-induced expression by real-time quantitative RT-PCR analysis. The experimental results also revealed that some genes may function in abscisic acid-dependent signaling pathway related to drought or salinity stress. Collectively, the data presented in this study will facilitate the understanding of molecular mechanism of B. napus in response to high-salinity and drought stresses, and also provide us the basis of effective genetic engineering strategies for improving stress tolerance of B. napus.

Modeling of protein refolding from inclusion bodies.

Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies (IBs), which becomes the major bottleneck in the preparation of recombinant proteins and their applications. In the present study, 36 proteins from IBs were refolded using a simple refolding method. Refolding yields of these proteins were defined as the percentage of soluble proteins following dilution refolding in the amount of denatured proteins in the samples before diluting into refolding buffer. Furthermore, a mathematical model was deduced to evaluate the role of biochemical properties in the protein refolding. Our results indicated that under the experimental conditions, isoelectric point of proteins might be mostly contributing to the high efficacy of protein refolding since the increment of one unit resulted in a decrease of 14.83% in the refolding yield. Other important mediators were components of protein secondary structure and the molecular weight (R(2) = 0.98, P = 0.000, F-test). Six proteins with low efficiency in the protein refolding possessed relatively low isoelectric points. Furthermore, refolding yields of six additional proteins from IBs were predicted and further validated by refolding the proteins under the same conditions. Therefore, the model of protein refolding developed here could be used to predict the refolding yields of proteins from IBs through a simple method. Our study will be suggestive to optimize the methods for protein refolding from IBs according to their intrinsic properties.

High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

BACKGROUND: Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. METHODS: We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. RESULTS: The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. CONCLUSION: Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

An automatic refolding apparatus for preparative-scale protein production.

Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production.

Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This "two-step-denaturing and refolding" (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.

[Application of a prediction model in inclusion body refolding].

OBJECTIVE: To establish a prediction method for the refolding of inclusion bodies and classify refolding types of different inclusion bodies directly from their primary structure to improve the efficiency of high throughput refolding process. METHODS: Forty-three recombinant proteins performing important biological functions were expressed in E. coli. The probability of forming inclusion bodies of these proteins was predicted using Harrison's two parameter prediction model based on the proteins' amino acid composition. Subsequently, the proteins from the inclusion bodies were refolded using a double denaturation method that involved washing and denaturation in GdnHCl solution followed by denaturation in Urea solution and refolding through dilution. RESULTS: All the proteins were detected in the form of inclusion bodies using SDS-PAGE method. The proteins were divided into two types according to the results of both solubility prediction and refolding experiments. Fourteen proteins were predicted to have the dependency of soluble expression. The refolding yields of these inclusion bodies were up to 70%. Twenty-nine proteins were predicted to have the high dependency of insoluble expression, and their refolding yields could be higher than 70% and lower than 60%. Comparison of the characteristics between the proteins with high and low refolding yields showed that the theoretical pI was significantly different (P<0.05). CONCLUSIONS: Harrison's two parameter prediction model has the value for potential application in classification of the inclusion bodies and prediction of solubility of proteins refolded from different inclusion bodies. This a novel method enhances the efficiency of high throughput refolding of inclusion bodies, and suggests that the theoretical pI of the proteins is an important parameter in the prediction of refolding yields.