ABSTRACT
In the research and development of new drugs, it is very important to investigate the in vitro metabolism of candidate drugs. Traditional models such as liver microsomes have many limitations, while the in vitro model of recombinant human drug metabolizing enzymes is considered as an important and useful approach because of its convenient access, stable activity and low cost. In this study, six major human UDP-glucuronosyltransferases (UGTs) genes (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) were cloned from human liver cDNA and heterologously expressed in Saccharomyces cerevisiae and baculovirus-infected insect cell. UGT1A1, 1A3, 1A6 and 1A9 were successfully expressed in yeast and showed glucuronidation activity against a variety of different structural types of substrates, but their activities were low. All six UGTs were successfully expressed and exhibited significantly improved glucuronidation activity when Trichopolusia ni cells BTI-TN5B1-4 (High Five) were used as the host. The recombinant human UGTs expressed in insect cells can catalyze the glucuronidation of their specific substrates, and the glucuronidation products were synthesized at milligram-scale with yields of 13%-66% for the first time, of which the structures were identified via MS, 1H NMR, and 13C NMR spectroscopic analysis. Above all, the recombinant human UGTs yeast and insect cell expression systems constructed in this study can be used for in vitro metabolism evaluation in the early stage of new drugs research and development, and also provide a new tool for the synthesis of glucuronide metabolites.
ABSTRACT
Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 ( HIV-1 ) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90％ and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.
ABSTRACT
Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 ( HIV-1 ) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90％ and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.
ABSTRACT
The baculovirus expression system has been used extensively for the expression of recombinant proteins in insect cells.Recently,recombinant baculoviruses containing mammalian cell-active promoter element have been used to transduce a broad spectrum of primary and established mammalian cells,including non-hepatic cells.Recombinant baculoviruses have been used successfully for transient or stable gene delivery in mammalian cells in vitro,while the efficiency of delivering gene in vivo is inhibited obviously by complements,but efforts have been made to overcome the problems,for instance,VSV-G-pseudotyped baculoviruses display complement resistance.The mechanism of the transduction is still not clearly understood,though many researches have been done.The baculovirus is able to replicate only in insect cells,but it is incapable of initiating replication cycle in mammalian cells,which guarantees high biosafety of this gene delivery system.In addition,this system is easily manipulated and able to carry large inserts.These attributes will undoubtedly lead to the increased application and continued development of this system for highly effective gene delivery into mammalian cells.