Production
and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in
NiCo21(DE3) Escherichia coli
Sin Yeang
Teow, Siti Aisyah Mualif, Tasyriq Che Omar, Chew Yik Wei, Narazah Mohd Yusoff
and Syed A Ali
Author
Affiliations
BMC
Biotechnology 2013, 13:107
doi:10.1186/1472-6750-13-107
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 [degree sign]C and
induction with 0.05mM 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. Enviado por bmc
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