To investigate the mechanism of high product specificity of gamma-clodextrin glucanotransferase (CGTase) from alkalophilic Bacillus clarkii 7364, we aligned protein sequence and structure model, found out that loss of 6 amino acids at subsite -7 probably affected its product specificity. Using overlapping PCR method, we inserted 6 amino acids into subsite -7 of CGTase. The mutant CGTase gene was ligated with pET-20b (+) and expressed in Escherichia coli BL21 (DE3). The extracellular recombinant enzyme was used to transform soluble starch into cyclodextrins (CDs). HPLC analysis results show that, compared to wild CGTase, the gamma-CDs produced by mutant enzyme decreased from 76.0% to 12.5%, whereas the ratio of alpha- and beta-CDs increased from 8.7% and 15.2% to 37.5% and 50%. The possible mechanism was that, compared to alpha-, beta-CGTase, wild gamma-CGTase lacks 6 amino acids in its subsite -7. This conformation provided more space for glucose combination and was thus advantageous for forming gamma-CD. When the 6 amino acids were inserted into the subsite -7 of wild gamma-CGTase, the space to bind with glucose reduced and consequently resulted in less gamma-CD production.
Bacillus ; enzymology ; genetics ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; biosynthesis ; genetics ; Mutant Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Apolipoprotein A-I-Milano(AIM), a natural variant, not only inhibits the initiation and progression of atherosclerosis, but also makes the preexisting atherosclerotic lesions regress. AIM gene, at which N-terminal codens were optimized, was subcloned into the expression vector of pET22b. Recombiant plasmids were transformed into E. coli strain BL21 (DE3) and induced with IPTG. The expressed apoliprotein A-I-Milano was soluble in E. coli and was about 38% of total cell lysate. Purified by Butyl Sepharose 4F. F hydrophobic chromatography and Q Sepharose H.P. anion exchange chromatography, followed by ultrafiltration with Vivaspin 20 (30 000MW), AIM monomer was obtained in a purity of more than 95%. Activity assay of binding of AIM monomer to lipid indicates that association of AIM monomer with DMPC is slower than normal apoA-I but DMPC number associated by AIM monomer is more than by apoA-I. This results will be important for studying structure, function of AIM, specially clinical application.
Apolipoprotein A-I ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Mutant Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Solubility

We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of alpha-cyclodextrin glucanotransferase (alpha-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as delta6). All these mutant alpha-CGTases were actively expressed in E. coli BL21. Compared with the wild-type alpha-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of alpha-CD was decreased from 68% to 30%, and beta-CD was raised from 22.2% to 33.3%. Interestingly, gamma-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and delta6 still produced alpha-CD as main product though the percentage of beta- and gamma-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type alpha-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce gamma-CD as the main product.
Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Mutation ; Paenibacillus ; enzymology ; Recombinant Proteins ; genetics ; gamma-Cyclodextrins ; metabolism

Heat shock factor 1 (HSF1) is the key protein in regulating stress response. It can be activated under heat, oxidative or another stress conditions. Dominant-positive and dominant-negative HSF1 are two types of HSF1 mutants. Both of them gain the DNA binding activity in the absence of stress. In addition, dominant-positive HSF1 acquires transcriptional activity, which dominant-negative HSF1 does not acquire. In this paper, the progress of using these HSF1 mutants in the research of cancer, neurodegenerative disorders and cardiovascular diseases will be discussed.
Genetic Therapy ; Heat-Shock Proteins ; genetics ; therapeutic use ; Humans ; Mutant Proteins ; genetics ; Neoplasms ; therapy ; Neurodegenerative Diseases ; therapy

A chimeric protein called Wallerian degeneration slow (Wld(S)) was first discovered in a spontaneous mutant strain of mice that exhibited delayed Wallerian degeneration. This provides a useful tool in elucidating the mechanisms of axon degeneration. Over-expression of Wld(S) attenuates the axon degeneration that is associated with several neurodegenerative disease models, suggesting a new logic for developing a potential protective strategy. At molecular level, although Wld(S) is a fusion protein, the nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) is required and sufficient for the protective effects of Wld(S), indicating a critical role of NAD biosynthesis and perhaps energy metabolism in axon degeneration. These findings challenge the proposed model in which axon degeneration is operated by an active programmed process and thus may have important implication in understanding the mechanisms of neurodegeneration. In this review, we will summarize these recent findings and discuss their relevance to the mechanisms of axon degeneration.
Animals ; Axons ; physiology ; Humans ; Mice ; Mice, Mutant Strains ; Models, Neurological ; Mutant Proteins ; genetics ; physiology ; Mutation ; NAD ; biosynthesis ; Nerve Degeneration ; etiology ; genetics ; physiopathology ; Nerve Tissue Proteins ; genetics ; physiology ; Nicotinamide-Nucleotide Adenylyltransferase ; genetics ; physiology

To increase the in vivo half-life of human CNTF mutein AX15 (R13K), HSA-AX15 (R13K) fusion protein was constructed by the fusion of the C-terminus of HSA to the N-terminus of AX15 (R13K) via an 11 amino acids linker. HSA-AX15 (R13K) fusion protein was purified to homogeneity by cation exchange chromatography, reverse phase chromatography and gel filtration after expressed in pichia pastoris. TF-1 cell survival bioassay showed the biological activity of AX15 (R13K) was not affected by the fusion to HSA. It was demonstrated that tertian injection of 4.8 mg/kg HSA-AX15 (R13K) fusion protein could produce more potent anti-obesity effects on KM mice than daily injection of 1.6 mg/kg AX15 (R13K). The long-acting form of hCNTF variant has the potential to reduce discomfort by requiring fewer injections and to minimize the side-effects by decreasing the dosage and fluctuation of plasma concentration, and thus has superior clinical application.
Animals ; Ciliary Neurotrophic Factor ; genetics ; Humans ; Mice ; Mutant Proteins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Serum Albumin ; genetics

OBJECTIVETo study the over-expression of mutant p53 protein in non-mucinous adenocarcinoma in-situ (NMAIS) and invasive adenocarcinoma, NOS of lung.

METHODSImmunohistochemical study for p53 protein was performed on 17 cases of NMAIS and 70 cases of invasive adenocarcinoma, NOS of lung. The difference in p53 over-expression between the two tumor subtypes was analyzed.

RESULTSThe over-expression of mutant p53 protein was observed in 0 case (0%) of NMAIS and 37 cases (52.9%) of invasive adenocarcinoma, NOS of lung. The difference was of statistical significance (P = 0.000).

CONCLUSIONMutant p53 protein over-expression may play a role in the progression of NMAIS to invasive adenocarcinoma, NOS.

Adenocarcinoma ; metabolism ; Adenocarcinoma in Situ ; metabolism ; Humans ; Immunohistochemistry ; Mutant Proteins ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism

Glucose-regulated protein 75 (Grp75) binds to p53 and inhibits its nuclear translocation, and thus plays a role in cell protection. To investigate whether the binding of Grp75 and p53 would influence the viability of cells, we constructed the eukaryotic expression vector of Grp75 deletion mutant. The deletion mutant gene was obtained by SOE-PCR (gene splicing by overlap extension) and then linked to the pcDNA3.0 vector. The constructed specific expression vector, pcDNA3.0/Grp75(Δ253-282), was identified by restriction enzymes and sequencing. Then we used liposome to transfect the specific vector into PC12 cells. The stable cell strain PC12/Grp75(Δ253-282)(+) was selected by G418 (1 mg/mL). Semi-quantitative RT-PCR and Western blot showed that Grp75 mRNA and protein expressions in PC12/Grp75(Δ253-282)(+) cells were higher than those in PC12 cells. The viability of cells undergoing 0 h, 3 h, 9 h, 18 h and 36 h of glucose deprivation respectively was measured by MTT assay. The results showed that the cell viability of PC12/Grp75(+) group was significantly higher than that of the other two groups, and the cell viability of PC12/Grp75(Δ253-282)(+) group was significantly higher than that of the PC12 group (P<0.05). Hoechst33324 staining was employed to detect cell apoptosis and the results were consistent with the MTT assay results. Western blot results indicated that the expression of p53 in PC12/Grp75(+) cells was lower than those in the other two groups, which might be due to the overexpression of Grp75. These results suggest that the protective role of Grp75 is partly associated with its binding to p53. The above results suggest that Grp75 deletion mutation could to some extent reduce the viability of cells.
Animals ; Gene Deletion ; Genetic Vectors ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; PC12 Cells ; Rats ; Transfection

Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.
Aminoacyltransferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biocatalysis ; Biotin ; Cysteine Endopeptidases ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genes, Reporter ; Ligation ; Mutant Proteins ; genetics ; metabolism

In the study, a gene encoding Tat protein N terminal 1- 21 amino acid residues-deleted mutant (Tat22-101) was amplified by PCR from a full length Tat gene of human immunodeficiency virus type 1, and the prokaryotic expression plasmid pET32a-Tat22-101 was constructed. After identification by digestion with endonucleases and sequencing, the recombinant plasmid pET32a-Tat22-101 was transformed into E. coli BL21(DE3) and expressed with IPTG induction. The mutant fusion protein with deleted Tat N terminal was purified by an affinity chromatography column Ni(2+)-NTA and subsequently identified by SDS-PAGE and Western blotting. The results showed that the molecular weight of the mutant protein was approximately 26.9kD. Furthermore, BALB/c mice were immunized with the mutant protein and the anti-sera were collected. ELISA results showed that the mutant protein preserved its immunogenicity, particularly it could improve the production of antibodies to other epitopes in addition to the N terminal epitope of Tat protein, which might provide some valuable information for the study of Tat functions as well as for development of potential novel HIV Tat vaccine.
Animals ; Female ; Gene Products, tat ; biosynthesis ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Mutant Proteins ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology