Zi-Chun Hua

Increased solubility of glutathione S‐transferase‐p16 (GST‐p16) fusion protein by co‐expression of chaperones groes and groel in Escherichia coli

Human cdk (cyclin dependent kinase) inhibitor p16 was fused with glutathione S‐transferase (GST) and the GST‐p16 fusion protein is under the control of T7 promoter. When expressed in E. coli BL21(DE3), most products existed in the form of insoluble inclusion bodies. When co‐expressed with molecular chaperones E. coli GroESL, most GST‐p16 products accumulated in the soluble form with a 5‐6 fold increase in solubility. When co‐produced with human protein disulfide isomerase (PDI), there was no improvement in the solubility of GST‐p16 fusion protein.

RENATURATION AND PURIFICATION OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR EXPRESSED IN E. COLI

Under the control of trp promoter, human tissue‐type plasminogen activator was expressed in E. coil in the form of inclusion body. The recombinant t‐PA was recovered for renaturation from preparative native PAGE gel by zinc acetate staining and electroelution. After renaturation in vitro, the recombinant t‐PA was purified by benzamidine affinity chromatography and lysine affinity chromatography. The purified t‐PA showed homogeneous on silver‐stained SDS‐PAGE gel, with a specific activity of 240,000I.U./mg protein.

Chemical conjugation of Gly‐Pro‐Arg‐Pro tetrapeptide to low molecular weight urokinase

Two low molecular weight urokinase derivatives were obtained by covalent coupling of a synthetic Gly‐Pro‐Arg‐Pro tetrapeptide to peptide A of low molecular weight urokinase and exchanging the native peptide A of low molecular weight urokinase with Gly‐Pro‐Arg‐Pro‐peptide A to obtain derivative I or direct conjugation of Gly‐Pro‐Arg‐Pro tetrapeptide to low molecular weight urokinase to obtain derivative II. In caseinolytic assay, fibrin can stimulate the two derivatives to activate plasminogen. But the two derivatives showed different kinetic behaviors. The derivative I displayed immediate onset of lysis and derivative II displayed a lag phase.

A low molecular weight urokinase derivative with enhanced fibrin affinity.

A Gly‐Pro‐Arg‐Pro tetrapeptide, homologous to amino‐terminal segment of the human fibrin α chain after the release of the fibrinopeptide A, was covalently coupled to peptide A of low molecular weight urokinase. The resulting derivative gained increased affinity for fibrin. In caseinolytic assay, fibrin can stimulate the derivative to activate plasminogen. The derivative had two‐fold greater fibrinolytic potency than native low molecular weight urokinase and its affinity for fibrin clot was 3.9‐fold higher than that of low molecular weight urokinase.

Quantum Yields in Photolyses of Mammalian Carboxy-Hemoglobin Studied by Pulsed Laser Pump-Probe Technique

The mechanisms describing the cooperative binding of CO or other ligands to hemoglobins (Hb) has been widely studied as an important fundamental problem for a long time. In this paper, the quantum yields in photolyses of carboxy-hemoglobins (HbCO) of mammals, such as human, pig, bovine, horse and rabbit, are investigated by the optical pump-probe technique. In the technique, the HbCO of the mammals are irradiated by a pulsed laser beam, and then the HbCO is photo-dissociated. Another continuous optical beam is used as a probe beam to detect the absorbance change induced by the photo-dissociation of the HbCO. The measured results show that the photolysis quantum yields of the HbCO are different for varies mammals, among them the quantum yields for porcine, bovine and equine HbCO are very close, but for the HbCO of rabbit, which is much smaller than the others. Finally, the results are analyzed and discussed.

Enhancement of in vitro renaturation of recombinant human pro‐urokinase by ampicillin

Ampicillin was used in vitro renaturation process of recombinant human prourokinase and it can obviously improve the yield of the renaturation, although not as well as arginine. When ampicillin was used together with arginine or lysine, it decreased the yield of the renaturation comparing with the yield when arginine or lysine used alone.

Influence of HbCO Structure of the Bar-Headed Goose on Photolysis Thermodynamics as Studied by the Nanosecond Laser-Ultrasonic Technique

The bar-headed goose, a specialized high-altitude species, has a capacity for high oxygen uptake from a hypoxic environment. It thus has a higher oxygen affinity than other bird species of lower-altitude environments. Oxygen affinity is determined by molecular structures and genetic mutations of hemoglobin (Hb), which can also influence the coordinating structures and dynamics of oxygen-Hb. To explore the structural differences in Hbs as between high and low altitude species, photolysis dynamic parameters, including quantum yield, enthalpy, and conformational volume changes in carboxy-Hbs (HbCO) for the bar-headed goose and low altitude counterparts (the Chinese goose and chicken) were investigated by the laser pumping-probing technique and photoacoustic calorimetry. Comparing the photolysis results for HbCO of the three species, the enthalpy and conformational volume changes of the bar-headed goose were much smaller than those of the others, although the quantum yields of all three species are similar. To explain the possible mechanisms of these differences, modifications of salt bridges and key residue mutations at the α β subunit interfaces of the proteins are described and discussed briefly.

Photolyses of carboxy-hemoglobin of bar-headed goose studied by photoacoustic calorimetry

As a specialized species native to high altitude, bar-headed goose can fly annually over an altitude of 9000m, which means that its hemoglobin has a higher oxygen affinity than its lowland relatives, such as goose and chicken. To study the mechanism of the phenomena, laser ultrasonic calorimetry is used to study dynamic processes associated with photolyses of carboxy hemoglobin (HbCO), including the enthalpy and conformational volume changes, of bar-headed goose and its lowland relatives. Considering the time scales of the reaction lifetimes in the photolyses processes of HbCO, two kinds of piezoelectric transducers, a PVDF film and a PZT ceramic, are used as acoustic signal detectors. For evaluating the relative enthalpy change and the relative conformational volume change in the process, the quantum yield of the photolysis must be taken into account, which has been measured by pump-probe technique. The results show that the enthalpy and conformational volume changes of bar-headed goose are obviously sma...