Optimization of recombinant human nerve growth factor production in the psychrophilic Pseudoalteromonas haloplanktis.
The optimization of production strategy is a very useful tool to attain high level of recombinant protein at a low cost. A promising biotechnological application of psychrophilic bacteria is their use as non-conventional host for the recombinant production of useful proteins. The lowering of the expression temperature can in fact facilitate the correct folding of heterologous proteins that accumulate in insoluble form as inclusion bodies when produced in Escherichia coli. An example of such "difficult" proteins is the human nerve growth factor (hNGF). The gene encoding the mature form of hNGF was expressed in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 at 4 degrees C. Western blotting experiments demonstrated that the protein was produced in soluble form and translocated in the periplasmic space. Furthermore, an analytical gel filtration chromatography confirmed that the recombinant protein was largely in dimeric form. For a more efficient recombinant rhNGF production, the influence of cultivation operational strategies and growth conditions (medium composition, temperature, specific growth rate) on biomass yield and recombinant protein production was investigated in batch and chemostat cultivations. The highest product yield of soluble rhNGF (7.5mg(NGF)g(dryweight)(-1)) has been achieved in batch culture at 4 degrees C on Schatz medium with addition of tryptone and vitamins.
Vigentini
Ileana
I
Università degli Studi di Milano, Dipartimento di Scienze Biomolecolari e Biotecnologie, Via Celoria, 26 20133 Milano, Italy.
Merico
Annamaria
A
Tutino
Maria Luisa
ML
Compagno
Concetta
C
Marino
Gennaro
G
D003470:Culture Media; D005285:Fermentation / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D020932:Nerve Growth Factor / Q000096:biosynthesis*; D031161:Pseudoalteromonas / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013696:Temperature
D003470:Culture Media; D011994:Recombinant Proteins; D020932:Nerve Growth Factor