[Genetic analysis of T1 progeny of transgenic tobacco with metallothionein gene and metallothionein domain mutant alpha alpha gene].

The chimeric gene containing a cloned mouse metallothionein processed gene or a cloned mouse metallothionein domain mutant alpha alpha gene was respectively introduced into tobacco (Nicotiana tobacum L. cv. NC89) on a disarmed Ti-plasmid of Agrobacterium tumefaciens. T1 seeds from self-fertilized transgenic tobacco were germinated on media containing cadmium or herbicide PPT. The PPT tolerance trait followed Mendelian inheritance and co-segregated with heavy metal tolerance. Meanwhile Southern blot and Western blot verified the existence of the MT gene and alpha alpha mutant gene in the T1 generation plants which keep tolerance to heavy metal. All the results demonstrated the stable integration and inheritance of exotic genes. In addition, assay of the root length and fresh weight of T1 seedlings indicate that transgenic tobacco plants with alpha alpha mutant gene still have a little higher tolerance than that with matural MT gene.

[Application of a gel chromatography renaturing way on low molecular single-chain urokinase mutant(DscuPA-32K)].

A recombinant mutant gene with thrombolytic and antithrombolytic bifunction was expressed in E. coli. Owing to two reasons of high molecular weight and over expression, dscuPA existed in inclusion body form. The protein of inclusion body was inactive protein. In order to obtain active protein, inclusion bodies should be denatured and then renatured. We performed a novel way named gel-chromatography column renaturation way. Compare with traditional renaturation way, this refolding approach had some obvious advantages, such as low cost and high recovery, and accomplished the preliminary purification step of desired protein(DscuPA-32K). Especially to proteins that easily became inactive and degradation, this approach might have good prospect.

[Studies on the expression condition of human trefoil factor 3 in Pichia pastoris].

In order to enhance the expression level of human trefoil factor 3 (hTFF3) in Pichia pastoris, we optimized the transformant growth conditions in shake flasks including carbon sources in growth medium, inoculation ratio, methanol concentration, pH rotation speed and inducing time. The transformant could grow on the glucose to OD600 5.0 after 14 hours inoculation. The best inoculation ration of 100 mL growth medium to the induction medium was 1:1. The expression level of dimeric human trefoil factor 3 induction with 1% methanol for 48 hours at pH 6.0, agitation speed 240 r/min could reach 20 mg/L with OD600 15. The protein was expressed in 5-liter fermentor with 2% methanol induction for 32 hours, finally the cell density reached OD600 120. 100 mg/L of recombinant hTFF3 was obtained in the supernatant.

Expression of Human Trefoil Factor 3 in Pichia pastoris and Its Biological Activity Analysis.

The human TFF3 (trefoil factor 3) DNA fragment was amplified by polymerase chain reaction (PCR) from human fetal placenta cDNA. The gene was cloned into the Pichia pastoris expression vector pPIC9K containing AOX 1 promoter and alpha-factor leader sequence. Multi-copies insertion transformants were screened on G418 plates. After the induction by 2% methanol for 48 hours, the expression of dimeric hTFF3 came up to 45% of total proteins in medium, as identified by SDS-PAGE and Western blot assay. The recombinant protein was further purified by S-Sepharose, Q-Sepharose ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography to the 95% purity, as shown by densitometric scanning. The N-terminus and molecular weight of the recombinant hTFF3 was in good agreement with the native hTFF3. The recombinant protein was proved to have good biological activity of preventing rats from the gastric ulcer induced by hydrochloric acid.

[Construction and characterization of the chimeric protein consisting RGD-containing peptide and proUK].

On the basis of structure analysis and computer modeling of proUK, two-chain DNA fragment encoding RGD peptide was inserted into the corresponding proUK cDNA site between Gly118-Leu119, using site-directed mutagenesis and DNA recombinant techniques. The chimeric gene was expressed in methylotrophic yeast Pichia pastoris expression system. The chimeric protein was purified after two step purification of Zn2+ chelating column and SP cation exchange column. The specific activity was 65,000 IU/mg protein. The chimeric protein had somewhat lower catalytic efficiency (kcat/Km) on the substrate S2444 as compared to Urokinase. But it had high anti-platelet aggregation activity, and the half inhibit constant was 2.1 mumol/L. The results showed that the chimeric protein not only had higher thrombolytic activity but also obtained anti-thrombus function. Further evaluation of the thrombolytic and antithrombolytic potential in appropriate animal models seemed to be investigated.

[Mixotrophic cultivation of Synechocystis sp. 6803: influences of light intensity and glucose].

The growth characteristics and the effects of glucose and light on the growth of Synechocystis sp. PCC6803 under mixotrophic conditions have been investigated. An exponential growth phase had been observed before glucose was consumed. The results showed that both of light intensity and glucose concentration had obvious effects on the mixotrophic growth of Synechocystis sp. PCC6803: the specific growth are decreased with the increase of glucose concentration at light; the specific growth rate and biomass/glucose yield increased with light intensity, ranging from 15-55 microE.m-2.s-1; when light intensity was higher than 55 microE.m-2.s-1, light saturation appeared.

Purification and characterization of an SDS-activated fibrinolytic enzyme from Eisenia fetida.

A sodium-dodecyl-sulfate-activated fibrinolytic enzyme from Eisenia fetida (the E. fetida enzyme) was purified by chromatography on DEAE Sepharose, Sephadex G-75, and Phenyl Sepharose 4. It (M(r) = 45 kDa) was composed of two subunits (M(r) = 26 kDa and M(r) = 18 kDa) held together by hydrophobic interactions. The enzyme displayed four activities when we used fibrin plates to detect the proteolytic activity. These were designated as CFPg (complete fibrinolysis in the plasminogen-rich plate), uCFPg (uncompleted fibrinolysis in the plasminogen-rich plate), CF (complete fibrinolysis in the plasminogen-free plate), and uCF (uncompleted fibrinolysis in the plasminogen-free plate). SDS activated CFPg and rendered the enzyme more sensitive to some inhibitors. Leupeptin, chymostatin, pepstatin, aprotinin, phenylmethylsulfonyl fluoride, and dithiothreitol had no effect on uCF. Pepstatin stimulated CFPg and uCFPg, while E-64, a thiol inhibitor, activated uCFPg and uCF. The N-terminal sequence of the large subunit was analyzed and compared with some known proteins. The large subunit alone had catalytic activity, while the small subunit did not. Using plasminogen as the substrate for defining peptide bond specificity, the E. fetida enzyme was observed to cleave the carboxyl side of basic amino acids, small neutral amino acids, and Met residue.

Fluorometric study of the isoform difference of mammalian metallothionein.

A fluorometric method for studying the isoform difference of mammalian metallothionein (MT), which lacks aromatic amino-acid residues, is reported. Cadmium-induced rabbit and hedgehog liver MT exhibited a strong luminescence signal in the range of 335 to 340 nm when excited at 285 nm in aqueous solution. The differences in emission intensity of the two major isoforms of MTs are significant. When titrated with chloride acid, which is believed to proton the metal-thiolate coordination bound to the -SH group in MT, a 10-nm red-shift property of the emission spectrum was observed, and the red-shift properties of the isoforms varied with the species. The observed fluorescence property of MT was considered to be the result of its polypeptide chains, which was confirmed by comparing the luminescence and absorption spectra during the titration of MT with diethylenetriaminepentaacetic acid. The new luminescence property of MT should be useful in studying the isoform and function difference of MT.

Determination and purification of metallothioneins by high performance liquid chromatography.

A rapid, reproducible and sensitive high performance liquid chromatography (HPLC) method for the determination and purification of metallothionein-I (MT-I) and metallothionein-II (MT-II) in mouse and rabbit livers has been developed. Methallothioneins (MTs) were separated by an HPLC anion exchange column, eluted through a linear gradient of Tris buffer and the peak containing MTs was determined by atomic absorption spectrophotometry. Furthermore, the content of MT-I or MT-II was calculated by protein peak area in a short time (about 20 min). The sample to be tested was homogenized, centrifuged and saturated by cadmium. MT-I and MT-II were eluted at 15.9 and 19.3 min, respectively. The following mouse liver cytosols were tested: controls, Cd-injected samples and 60Co-irradiated samples. A detection limit of 5 micrograms/g liver was established for this method. We have analysed more than 100 biological samples and obtained satisfactory results.

Preparations and properties of crystalline porcine and bovine [Trp]B1 insulins.

[Trp]B1 analogs to porcine and bovine insulins have been obtained through selective reaction with Msc-ONSu, and followed by Edman degradation, condensation with Msc-Trp-ONp and deprotection. Through cellulose acetate electrophoresis, amino acid composition analysis, UV absorption spectrum and N-terminal analysis, it has been proved that the products are of purified [Trp]B1 insulin. Mouse convulsion assay shows that the biological activities of porcine and bovine [Trp]B1 insulins are 18 i.u./mg and 17 i.u./mg respectively, corresponding to about 70% of the original activity of native porcine insulin. Their structural details are under investigation in our laboratory.

Active products of porcine trypsin after autolysis.

Porcine trypsin obtained from pancreas residues subsequent to insulin removal undergoes autolysis when subjected to chromatography and gives rise to new forms of autolyzed products with intra-chain split at bonds Lys145-Ala146 and Arg105-Val106. Incubation of 1% solutions of porcine trypsin either at pH 5.0 or at pH 9.1 induces autolysis to give active products involving one or two specific cleavages of bonds Lys145-Ala146 and Arg105-Val106 or Lys131-Ser132, as well as inactive degraded products. No evidence has been obtained that on autolysis of porcine trypsin, and active fragment with molecular weight lower than that of the parent molecule was identified. The active forms of autolyzed products of porcine trypsin have almost the same specific activity as the intact enzyme when assayed against BAEE. They are of the same molecular weight as the parent molecule. These findings indicate that that active forms of autolyzed products maintain the specific three-dimensional structure essential for the catalytic activity of the trypsin molecule.