从大肠杆菌包含体中提取有活性的人溶菌酶的研究

从大肠杆菌包含体中提取有活性的人溶菌酶的研究钱世钧陈欣叶军郭良栋郭伟（中国科学院微生物研究所北京１０００８０）在医药和工业上，许多非常有应用前景的真核多肽，往往由于它们天然来源缺乏，不能充分提供产品，限制了它们的应用范围。通过基因克隆技术使其在大肠...

Renaturalizing Floodplains

This manuscript analyzes and discusses viewpoints concerning the renaturalization of floodplains as an instrument of management in large catchments, using natural flood defense schemes. Schemes consider the differentiated supply of ecosystemic services based on river channel/floodplain interactions. Conventional structural methods used to prevent flooding (e.g., longitudinal dikes) are increasingly showing themselves to be less efficient with regard to advances in the problems of environmental management of the territory, especially when combined with extreme events, where the importance of perfecting strategies for harmonizing duly controlled floodable areas and water retention can be seen. Natural flood risk reduction measures are part of a holistic solution for sustainable management of flood risk, conservation of nature, water quality and green economy. They rely upon the inherent ability of floodplains to retain water in the basin, and this can delay and reduce peak flows.

Studies on the Renaturation with Simultaneous Purification of Recombinant Human Proinsulin with Unit of Simultaneous Renaturation and Purification of Protein in Semi-preparative Scale

The renaturation and purification of recombinant human proinsulin (rh-proinsulin) expressed in E. coli with the unit of simultaneous renaturation and purification of protein (USRPP) in semi-preparative scale was studied. The result shows that rh-proinsulin extracted with 8.0 mol/L urea can be renatured and purified simultaneously in 45 minutes with the USRPP (1050 mm ID). The purity of rh-proinsulin was found to be more than 90% and the mass recovery to be more than 80%. The renaturation effect of rh-proinsulin with the USRPP was tested by enzyme cleavage for obtaining insulin. In addition, the result was further confirmed with RPLC, SDS-PAGE electrophoresis, and MALDI-TOF, respectively.

A Novel Method for Diminishing Protein Aggregation during Denatuaration Process

The addition of packing material for high performance hydrophobic interaction chromatograghy （HPHIC） into the denaturant solution to prevent, or depress protein aggregation in the denatuaration process is presented. The renaturation of α-chymotrypsin （α-Chy） denatured with guanidine hydrochloride （GuHCl） solution indicated that renaturation efficiency can be enhanced from 36.1% to 59.0% by this new method. The structure of the ligand linking of HPHIC packings is also important for the protein renaturation.

Prokaryotic expression and purification of fibronectin leucine rich transmembrane protein 3 C-terminal domain proteins in rats

BACKGROUND： Studies have suggested that fibronectin leucine-rich transmembrane protein 3 （FLRT3） is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE： To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins. DESIGN, TIME AND SETTING： An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008. MATERIALS： Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coil （E. coil） JM109 were purchased from Promega. E. coil BL21 was provided by Novagen. METHODS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. coil BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained. MAIN OUTCOME MEASURES： Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. The recombinant protein was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing. CONCLUSION： From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E. coil BL21. Moreover, highly purified GST fusion proteins were obtained.

Comparison of the Contributions of Tetrahydrofurfuryl Alcohol and PEG to a-Chymotrypsin Renaturation with High Performance Hydrophobic Interaction Chromatography

The contributions of tetrahydrofurfuryl alcohol (THFA) and polyethylene glycol (PEG) to the renatured efficiency of a-chymotrypsin were investigated and compared with each other. The maximum increments of bioactivity recovery of a-Chy were found to be 25.1% for THFA, 10.4% for PEG, respectively. The experimental results indicated that the denaturant solution containing THFA contributed more to the renaturation of a-Chy in high performance hydrophobic interaction chromatography (HPHIC) than that containing PEG, when the concentration of THFA was 3.2%, the bioactivity recovery of a-Chy is the highest.

Study of renaturation condition of anti-TNF-α recombinant antibodies

Objective: To study the rules in the renaturation of recombinant antibody. Methods: Anti-TNF-a single domain (Sd) and single chain fragment variety(SCFv) antibodies were separated and purified under four conditions, including cosolvent redoxing, surface active solvent inducting, denaturant solvent inducting and the antigen of rhu TNFL-a inducting. Results: The dissolubility of renaturation products were between 6% - 11 %. Conclusion: These several conditions were good enough to the 2 antibody proteins and the best in them is the combination of the antigen inducting with affinity chromatography.

Structure-activity correlationship and folding of recombinant Escherichia coli dihydro folate reductase (DHFR) enzyme through biochemical and biophysical approaches

The design of any antagonist or inhibitor for any enzyme requires the knowledge of structure- function relationship of the protein and the optimum conformational states for maximum and minimum activities. Furthermore, designing of the inhibitors or drugs against an enzyme becomes easier if there is information available about various well characterized intermediate conformation of the molecule. In vivo folding pathway of any recombinant protein is an important parameter for understanding its ability to fold by itself inside the cell, which always dictates the downstream processing for the purification. In the present manuscript we have discussed about the in vivo and in vitro folding, and structure-function relationship of Dihydrofolate reductase enzyme. This is an important enzyme involved in the cell growth and hence inhibition or inactivation of the enzyme may reduce the cell growth. It was observed that the equilibrium unfolding transition of DHFR proceeds through the formation of intermediates having higher exposed surface hydrophobicity, unchanged enzymatic activity and minimum changes in the secondary structural elements. Because of enhanced surface hydrophobicity, and unchanged enzymatic activity, these intermediates could be a nice target for designing drugs against DHFR.

In vitro renaturation of recombinant human pro-urokinase expressed in Escherichia coli

Objective Recombinant human pro-urokinase forms insoluble inclusion body when overexpressed in Escherichia coli. It must be denatured and renatured in vitro so that it can acquire activity. This study aimed at increasing the renaturation yield of denaturant pro-urokinase. Methods We evaluated the basic renaturation conditions of pro-urokinase through qualitative and quantitative analysis of pH, temperature, denatured concentration, protein concentration, and the ratio of reduced and oxidized thiol reagents. We also compared the effects of nonspecific additives, step-wise dilution and urea gradient dialysis.Results We defined the optimal conditions of pro-urokinase renaturation with a yield of about 20%-30%. Conclusion Different recombinant denatured proteins have different renaturation conditions due to their different molecular sizes, molecular constructions, disulfide bond numbers, and hydrophobicity. The renaturation yield can be increased by optimizing the renaturation conditions of a specific protein.

Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta （DE3） and induced by 1 mM IPTG for 4 h at 37℃. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni^2＋-affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues.

Expression of human transforming growth factor β1 in E. coli (I) --Direct expression in cytoplasm

PCR technique is used to amplify the mature peptide gene of human transforming growth factor β1(hTGFβ1); the gene is verified by full length sequence analysis. In DH5α/pBV220 expression system, hTGFβ1 attains expression in the cytoplasm of \%E. coli\% up to 16%. The recombinant protein is proved to be the monomer of hTGFβ1 by N terminal amino acids analysis and immunoblotting. After refolding of the monomer protein \%in vitro\% in glutathione system or CHPAS/DMSO system, the dimeric protein accumulates to 30% in the refolding mixture. The recombinant protein is purified to homogeneity on silver staining, and is shown to have strong biological activity from MTT bioassay on Mv1Lu cells.

Effect of Denaturant Concentration on Hen-egg White Lysozyme Renaturation

Based on three-state renaturation process of denatured proteins, an equation describing the effect of denaturant concentration on renaturation yield of denatured proteins was presented. By this equation, two parameters n（m1 -m2） and Ka can be obtained. The former indicates the difference in the number of denaturant molecules between the renaturation process of n number of refolding intermediates from refolding intermediate state to native state and their aggregate process from refolding intermediate state to aggregate state, the latter denotes the apparent aggregate equilibrium constant for protein molecules aggregated from native state to aggregate state, and from them, the characteristics of the renaturation process of denatured proteins in denaturant solution can be identified. This equation was tested by the renaturation processes of denatured egg white lysozyme in guanidine hydrochloride and urea solutions, with the results to show that when guanidine hydrochloride and urea concentrations were separately higher than 1.25 and 3.00 mol/L or separately lower than 1.00 and 3.00 mol/L, the refolding intermediates of egg white lysozymes were more easily aggregated to aggregate state or more easily renatured to native state, respectively. Under different initial total egg white lysozyme concentrations in urea solution, the refolding egg white lysozyme intermediates could be deduced to have a tendency to form a bimolecular intermediate aggregate, and this inference was further confirmed by their nonreducing SDS-PAGE and size exclusion chromatography.

Effect of ligand structure of stationary phase of high performance hydrophobic interaction chromatography on renaturation efficiency of GuHCl-denaturedα-chymotrypsin

The renaturation of the denaturedα-chymotrypsin(α-Chy)with 1.7 mol·L^(-1)guanidine hydrochloride(GuHCI)by three kinds of stationary phase of high performance hydrophobic interaction chromatography(STHIC)with a comparable hydrophobicity but different ligand structures was investigated.The obtained result indicates that the ligand structures of the three STHIC contribute to the renaturation efficiency ofα-Chy in the order of the end ligands PEG-600<phenyl group<tetrahydrofurfuryl alcohol(THFA).