title: 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.
  
  
  RESULTS
  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.
authors.forename: Sin Yeang
authors.initials: SY
authors.lastname: Teow
authors.forename: Siti Aisyah
authors.initials: SA
authors.lastname: Mualif
authors.forename: Tasyriq Che
authors.initials: TC
authors.lastname: Omar
authors.forename: Chew Yik
authors.initials: CY
authors.lastname: Wei
authors.forename: Narazah Mohd
authors.initials: NM
authors.lastname: Yusoff
authors.affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas, Pulau Pinang, Malaysia. ali2@amdi.usm.edu.my.
authors.forename: Syed A
authors.initials: SA
authors.lastname: Ali
mesh_terms: D036022:Capsid Proteins / Q000096:biosynthesis* / Q000235:genetics; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D016655:HIV Core Protein p24 / Q000096:biosynthesis* / Q000235:genetics; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D010957:Plasmids / Q000235:genetics; D058105:Polymerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins; D003470:Culture Media; D016655:HIV Core Protein p24; D011994:Recombinant Proteins; C514799:p24 protein, Human Immunodeficiency Virus Type 1