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Title
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Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris.
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Authors
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S.Goda,
K.Takano,
Y.Yamagata,
Y.Katakura,
K.Yutani.
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Ref.
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Protein Eng, 2000,
13,
299-307.
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PubMed id
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Abstract
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A human lysozyme expression system by Pichia pastoris was constructed with the
expression vector of pPIC9, which contains the alpha-factor signal peptide known
for high secretion efficiency. P. pastoris expressed the human lysozyme at about
300 mg/l broth, but four extra residues (Glu(-4)-Ala(-3)-Glu(-2)-Ala(-1)-) were
added at the N-terminus of the expressed protein (EAEA-lysozyme). To determine
the effect of the four extra residues on the stability, structures and folding
of the protein, calorimetry, X-ray crystal analysis and GuHCl denaturation
experiments were performed. The calorimetric studies showed that the
EAEA-lysozyme was destabilized by 9.6 kJ/mol at pH 2.7 compared with the
wild-type protein, mainly caused by the substantial decrease in the enthalpy
change (DeltaH). On the basis of structural information on the EAEA-lysozyme,
thermodynamic analyses show that (1) the addition of the four residues slightly
affected the conformation in other parts far from the N-terminus, (2) the large
decrease in the enthalpy change due to the conformational changes would be
almost compensated by the decrease in the entropy change and (3) the decrease in
the Gibbs energy change between the EAEA and wild-type human lysozymes could be
explained by the summation of each Gibbs energy change contributing to the
stabilizing factors concerning the extra residues.
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