Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris

A human lysozyme expression system by Pichia pastoris was constructed with the expression vector of pPIC9, which contains the a-factor signal peptide known for high secretion efficiency. P.pastoris expressed the human lysozym e at about 300 mg/I broth, but four extra residues (Glu(-4)-Ala(-3)-Glu(-2) -Ala(-1)-) were added at the N-terminus of the expressed protein (EAEA-lyso zyme), 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 studie s showed that the EAEA-lysozyme was destabilized by 9.6 kJ/mol at pH 2.7 co mpared with the wild-type protein, mainly caused by the substantial decreas e in the enthalpy change (Delta H). 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 fro m 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 e ntropy change and (3) the decrease in the Gibbs energy change between the E AEA and wild-type human lysozymes could be explained by the summation of ea ch Gibbs energy change contributing to the stabilizing factors concerning t he extra residues.