Recombinant production of anti-HIV protein, griffithsin, by auto-induction in a fermentor culture.
Griffithsin (GRFT) is a novel anti-HIV protein isolated from the red alga Griffithia sp. The potent anti-viral activity of GRFT against both laboratory and primary isolates of HIV at picomolar concentrations makes this protein an attractive candidate microbicide to prevent sexual transmission of HIV. Here, we describe the recombinant production and purification of a biologically active hexa-histidine-tagged GRFT (His-GRFT) from Escherichia coli. To facilitate a large-scale production of recombinant His-GRFT, we tested different expression conditions to optimize the expression in the cytoplasm of E. coli to increase the overall production of soluble His-GRFT. Attempts to express His-GRFT in shake flask cultures resulted in a modest yield of soluble His-GRFT, with a large accumulation of the protein in inclusion bodies. The use of a fermenter and of a rich, auto-inducing medium allowed the total amount of His-GRFT per liter to be increased by about 45-fold, with approximately 70% of the protein expressed in the soluble fraction. N-terminal sequencing and MALDI-TOF analyses of the recombinant His-GRFT confirmed that the initial methionine residue was cleaved off. Recombinant His-GRFT showed equivalent activity with natural GRFT, both in respect to gp120-binding characteristics as well as anti-HIV activity. Size-exclusion chromatography analysis showed that both native GRFT and recombinant His-GRFT existed as homodimers in solution. The expression system described in this work provides a basis for the mass production of GRFT to allow further studies of the protein and investigation of therapeutic and preventive strategies against HIV.
Giomarelli
Barbara
B
Molecular Targets Development Program, Center for Cancer Research, National Cancer Institute, NCI-Frederick, Frederick, MD 21702-1201, USA.
Schumacher
Kathryn M
KM
Taylor
Troy E
TE
Sowder
Raymond C
RC
Hartley
James L
JL
McMahon
James B
JB
Mori
Toshiyuki
T
D020418:Algal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D000595:Amino Acid Sequence; D019149:Bioreactors; D003001:Cloning, Molecular / Q000379:methods; D015658:HIV Infections / Q000517:prevention & control*; D037102:Lectins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D037121:Plant Lectins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D000461:Rhodophyta / Q000737:chemistry* / Q000502:physiology*; D012995:Solubility; D038301:Virus Inactivation
D020418:Algal Proteins; D037102:Lectins; D037121:Plant Lectins; D011994:Recombinant Proteins; C499654:griffithsin protein, Griffithsia