Letno poročilo 2005

zijskimi telesi smo za ta bolj topna inkluzijska
telesa, iz katerih lahko z blago ekstrakcijo
pridobimo aktivno obliko tar~nega proteina,
uvedli pojem »neklasi~na« inkluzijska telesa.
Medtem ko obi~ajen na~in pridobivanja
heterolognih proteinov iz inkluzijskih teles E.
coli navadno vklju~uje uporabo mo~nih
denaturantov in reducentov, ~emur sledi bolj
ali manj uspe{na faza renaturacije predhodno
popolnoma denaturiranega proteina, lahko
ekstrakcijo proteina iz »neklasi~nih« inkluzijskih
teles izvedemo v nedenaturirajo~ih
pogojih. Raztapljanje »neklasi~nih« inkluzijskih
teles poteka v primernih pufrih, ob
dodatku nizkih koncentracij blagih detergentov
ali polarnih topil oziroma nedetergentnih
sulfobetainov, pri ~emer ekstrahiramo
do 45% celokupno prisotnega
proteina v biolo{ko aktivni obliki. Prisotnost
pravilno zvitega proteina G-CSF v inkluzijskih
telesih, nastalih pod pogoji sinteze pri nizkih
temperaturah, smo potrdili z meritvami
biolo{ke aktivnosti in FT-IR spektralno analizo,
s poskusi na drugih proteinih (GFP, TNFβ in
njegovi analogi, specialni histidinski analogi
TNFα s histidinskim podalj{kom, pripetim na
skraj{an N-konec molekule) pa smo dokazali,
da je koncept »neklasi~nih« inkluzijskih teles
bolj splo{en in verjetno uporaben tudi za
enostavno pridobivanje drugih proteinov.
- Na primeru treh razli~nih oblik dejavnika
tumorske nekroze beta (TNFβ) smo preu~evali
vpliv strukture in dol`ine N-terminalnega dela
molekule na fizikalno-kemijske lastnosti
proteinov. V bakteriji E. coli smo pripravili
MetTNFβ, ki je slu`il kot primerjalna substanca
z naravno strukturo, in dva analoga TNFβ,
ki imata nekoliko spremenjen N-terminalni
del molekule. His7TNFβ je dalj{a oblika
proteina s heptahistidinskim podalj{kom,
pripetim na naravni N-konec, medtem ko
ima ∆N19TNFβ skraj{an N-terminalni del
molekule. Podobno kot v predhodnih
raziskavah strukturno sorodnega proteina
TNFα, smo ugotovili, da lahko s pripenjanjem
histidinskih podalj{kov na hidrofobne regije
Laboratorij za biosintezo in biotransformacijo
Laboratory for Biosynthesis and Biotransformation
ture leads to the formation of interesting
protein aggregates, containing a high
amount of properly folded target protein (or
its precursor). These inclusion bodies are more
fragile and more soluble than “classical” ones,
thus a new term “nonclassical” inclusion
bodies was proposed. While protein isolation
from “classical” inclusion bodies requires a
series of denaturation / renaturation steps
with relative low efficiency, higher solubility
and fragility of “nonclassical” inclusion bodies
enables extraction of biologically active
target protein by mild extraction under nondenaturating
conditions. “Nonclassical” inclusion
bodies are solubilized in adequate
buffers containing low concentration of mild
detergents, polar solubilisers or non-detergent
sulfobetains, and up to 45% of total
target protein is extracted in biologically active
form. Presence of correctly folded G-CSF
inside “nonclassical” inclusion bodies was
confirmed by FT-IR spectral analysis as well
as with biological activity determinations.
Studies on other proteins (GFP, TNFβ and its
analogs, analogs of TNFα with histidine tag
on N-terminus) confirmed that the concept
of “nonclassical” inclusion bodies is more
general and thus useful for simple cost-effective
downstream processes in biotechnology.
- We studied the relationship between the Nterminal
protein structure and its physicochemical
properties using three different
forms of tumor necrosis factor beta (TNFβ).
All three proteins were designed and expressed
in E.coli. MetTNFβ represented the
reference substance, His7TNFβ was the
longer form bearing a hepta-histidine tag
attached to the natural N-terminus, and
∆N19TNFβ possessed a shortened N-terminal
part. Similarly as in the case of the structurally
related protein TNFα, we concluded
that attachment of His patches to the hydrophobic
regions of the protein affects its solubility
and consequently distribution of the
protein between the soluble and insoluble
fraction of the cell.
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