A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli.

An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time from the Escherichia coli expression system using short Strep-tag II (ST II). The recHPL-ST II was solubilized using 8 M urea from E.coli lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses in the presence of glycerol and Ca2+ for 2 days followed by gel filtration, 1.8-6 mg of active recHPL-ST II was obtained from 1 L of culture. The recHPL was non-glycosylated, but showed almost equal specific activity, pH-dependency and time-dependent stability compared to those of native porcine pancreatic lipase (PPL) at 37°C. However, the recHPL lost its lipolytic activity above 50°C, showing a lower heat-stability than that of native PPL, which retained half its activity at this temperature.

Molecular cloning and expression of the pyranose 2-oxidase cDNA from Trametes ochracea MB49 in Escherichia coli.

A cDNA of a structural gene encoding pyranose 2-oxidase (P2O) from Trametes ochracea strain MB49 was cloned into Escherichia coli strain BL21(DE3) on a multicopy plasmid under the control of the trc promoter. Synthesis of P2O was studied in batch cultures in LB or M9-based mineral medium at 28 degrees C. While there was a low specific activity of P2O in LB medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 U mg(-1)). The effect of isopropyl beta-D-thiogalactoside on the expression of P2O was studied in mineral medium at 25 and 28 degrees C. The synthesis of P2O at 28 degrees C corresponded to 39% of the cell soluble protein but the major portion of P2O (93%) was in the form of non-active inclusion bodies (activity of P2O equalled 0.19 U mg(-1)). At 25 degrees C, the amount of P2O represented 14% of the cell soluble protein and the activity of P2O was 1.1 U mg(-1). The soluble enzyme represented 70% of the total amount of P2O.

Investigation of refolding condition for Pseudomonas fluorescens lipase by response surface methodology.

Response Surface Methodology (RSM) was used to investigate optimal refolding conditions of Pseudomonas fluorescens lipase which was expressed as inclusion body in E. coli. Three interacting factors, protein concentration, pH and guanidine hydrochloride (GdnHCl) concentration were selected as the variables of RSM. The protein concentration did not affect the refolding yield within the selected range (50-340 micrograms ml-1) at low temperatures of 4 and -15 degrees C, but it was a critical factor at 25 degrees C and refolding yield significantly decreased with increasing protein concentration. The pH and GdnHCl were significant factors in all experimental conditions. But there was no trends of optimal pH depending on temperature and additives, just showing the optimum at neutral pH. Glycerol shifted optimal GdnHCl concentration to higher side, while arginine to lower side. Therefore, it was concluded that glycerol and arginine deprives and supplements GdnHCl, respectively. In this study, RSM made it possible to investigate successfully the optimal conditions of in vitro refolding and to elucidate interactions between refolding factors with a minimum number of experiments. Under the optimal condition determined by RSM, approximately 90% refolding yield was obtained and it was a 30% increase over the conventional method.

Catalytic properties of murine carbonic anhydrase IV.

A cDNA encoding the murine carbonic anhydrase IV (mCA IV) gene, modified to resemble a form of mature human carbonic anhydrase IV (Okuyama, T., Waheed, A., Kusumoto, W., Zhu, X. L., and Sly, W. S. (1995) Arch. Biochem. Biophys. 320, 315-322), was expressed in Escherichia coli. Inactive inclusion bodies were collected and refolded, and active enzyme was purified; the resulting mCA IV was used to characterize the catalysis of CO2 hydration using stopped flow spectrophotometry and 18O exchange between CO2 and water. Unlike previously studied isozymes in this class of carbonic anhydrase, the pH profile for kcat for hydration of CO2 catalyzed by mCA IV could not be described by a single ionization, suggesting multiple proton transfer pathways between the zinc-bound water molecule and solution. A role for His64 in transferring protons between the zinc-bound water and solution was confirmed by the 100-fold lower activity of the mutant of mCA IV containing the replacement His64 --> Ala. The remaining activity in this mutant at pH levels near 9 suggested a second proton shuttle mechanism. The maximal turnover number kcat for hydration of CO2 catalyzed by mCA IV was 1.1 x 10(6) s-1 at pH > 9. A pKa of 6.6 was estimated for the zinc-bound water molecule in mCA IV.

Purification of an indole alkaloid biosynthetic enzyme, strictosidine synthase, from a recombinant strain of Escherichia coli.

The gene for the indole alkaloid biosynthetic enzyme, strictosidine synthase, of Catharanthus roseus has been cloned into an inducible Escherichia coli expression vector using an expression cassette polymerase chain reaction technique. Induction of the gene resulted in overexpression of the enzyme which accumulated mainly as insoluble inclusion bodies. Denaturation and refolding of the insoluble protein resulted in the ability to purify up to 6 mg of active enzyme from a single liter of cell culture. The recombinant enzyme has good activity (approximately 30 nkat/mg).