Dataset Information

Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli.

ABSTRACT: BACKGROUND: HIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and reproducible process for efficient production of HIV-1 CA. RESULT: In this work, 6-His-tagged wild type CA from HIV-1 (NL4.3) was expressed in rare tRNA-supplemented NiCo21(DE3) Escherichia coli, and its production was studied in shake flask culture condition of expression. Influences of various key cultivation parameters were examined to identify optimal conditions for HIV-1 CA production. It was found that a culture temperature of 22°C and induction with 0.05 mM IPTG at the early stage of growth were ideal, leading to a maximum biomass yield when grown in Super broth supplemented with 1% glucose. With optimized culture conditions, a final biomass concentration of ~27.7 g L?¹ (based on optical density) was obtained in 12 hours post-induction, leading to a yield of about ~170 mg L?¹ HIV-1 CA. A two-step purification strategy (chitin beads?+?IMAC) was employed, which efficiently removed metal affinity resin-binding bacterial proteins that contaminate recombinant His-tagged protein preparation, and resulted in highly pure HIV-1 CA. The purified protein was capable of polymerization when tested in an in vitro polymerization assay. CONCLUSIONS: By using this optimized expression and purification procedure, milligram amounts of highly pure and polymerization-competent recombinant HIV-1 CA can be produced at the lab-scale and thus used for further biochemical studies.

SUBMITTER: Teow SY

PROVIDER: S-EPMC4235032 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli.

Teow Sin Yeang SY Mualif Siti Aisyah SA Omar Tasyriq Che TC Wei Chew Yik CY Yusoff Narazah Mohd NM Ali Syed A SA

BMC biotechnology 20131204

<h4>Background</h4>HIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and ...[more]

PMID: 24304876

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Project description:2'-fucosyllactose (2'-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2'-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2'-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2'-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2'-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2'-FL production, ultimately a highest titer of 2'-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2'-FL was achieved with a yield of 0.89 mol 2'-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2'-FL. As a proof of concept, our preliminary 2'-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

Dataset Information

Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli.

Publications

Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli.

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