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


Abstract

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. Study holds ProTherm entries: 8069, 8070, 8071, 8072, 8073, 8074, 8075, 8076, 8077, 8078, 8079, 8080, 8081, 8082, 8083 Extra Details: four extra residues (Glu-4-Ala-3-Glu-2-Ala-1-) were added at,the N-terminus of the expressed protein (EAEA-lysozyme) crystal structure; extra N-terminal residues; human lysozyme;,Pichia pastoris; protein stability

Submission Details

ID: kVt8oa4H4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Goda S;Takano K;Yamagata Y;Katakura Y;Yutani K,Protein Eng. (2000) Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris. PMID:10810162
Additional Information

Study Summary

Number of data points 54
Proteins Lysozyme C ; Lysozyme C
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Cm buffers:Sodium phosphate: 50 mM, pH:7.0 ; Experimental Assay: m buffers:Sodium phosphate: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O buffers:Sodium phosphate: 50 mM, pH:7.0 ; Experimental Assay: Cm pH:4.0, buffers:glycine-HCl: 50 mM ; Experimental Assay: m pH:4.0, buffers:glycine-HCl: 50 mM ; Experimental Assay: dG_H2O pH:4.0, buffers:glycine-HCl: 50 mM ; Experimental Assay: Cm pH:3.0, buffers:glycine-HCl: 50 mM ; Experimental Assay: m pH:3.0, buffers:glycine-HCl: 50 mM ; Experimental Assay: dG_H2O pH:3.0, buffers:glycine-HCl: 50 mM ; Experimental Assay: dCp pH:3.03 ; Experimental Assay: dHcal pH:3.03 ; Experimental Assay: Tm pH:3.03 ; Experimental Assay: dHvH pH:3.03 ; Experimental Assay: dCp pH:2.93 ; Experimental Assay: dHcal pH:2.93 ; Experimental Assay: Tm pH:2.93 ; Experimental Assay: dHvH pH:2.93 ; Experimental Assay: dCp pH:2.87 ; Experimental Assay: dHcal pH:2.87 ; Experimental Assay: Tm pH:2.87 ; Experimental Assay: dHvH pH:2.87 ; Experimental Assay: dCp pH:2.84 ; Experimental Assay: dHcal pH:2.84 ; Experimental Assay: Tm pH:2.84 ; Experimental Assay: dHvH pH:2.84 ; Experimental Assay: dCp pH:2.78 ; Experimental Assay: dHcal pH:2.78 ; Experimental Assay: Tm pH:2.78 ; Experimental Assay: dHvH pH:2.78 ; Experimental Assay: dCp pH:2.75 ; Experimental Assay: dHcal pH:2.75 ; Experimental Assay: Tm pH:2.75 ; Experimental Assay: dHvH pH:2.75 ; Experimental Assay: dCp pH:2.68 ; Experimental Assay: dHcal pH:2.68 ; Experimental Assay: Tm pH:2.68 ; Experimental Assay: dHvH pH:2.68 ; Experimental Assay: dCp pH:2.64 ; Experimental Assay: dHcal pH:2.64 ; Experimental Assay: Tm pH:2.64 ; Experimental Assay: dHvH pH:2.64 ; Experimental Assay: dCp pH:2.63 ; Experimental Assay: dHcal pH:2.63 ; Experimental Assay: Tm pH:2.63 ; Experimental Assay: dHvH pH:2.63
Libraries Mutations for sequence KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
133L 1993-06-01T00:00:00+0000 1.77 ROLE OF ARG 115 IN THE CATALYTIC ACTION OF HUMAN LYSOZYME. X-RAY STRUCTURE OF HIS 115 AND GLU 115 MUTANTS
134L 1993-06-01T00:00:00+0000 1.77 ROLE OF ARG 115 IN THE CATALYTIC ACTION OF HUMAN LYSOZYME. X-RAY STRUCTURE OF HIS 115 AND GLU 115 MUTANTS
1B5U 1999-01-11T00:00:00+0000 1.8 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANT
1B5V 1999-01-11T00:00:00+0000 2.17 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANTS
1B5W 1999-01-11T00:00:00+0000 2.17 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANTS
1B5X 1999-01-11T00:00:00+0000 2.0 Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and x-ray analysis of six ser->ala mutants
1B5Y 1999-01-11T00:00:00+0000 2.2 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANTS
1B5Z 1999-01-11T00:00:00+0000 2.2 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANTS
1B7L 1999-01-24T00:00:00+0000 1.8 VERIFICATION OF SPMP USING MUTANT HUMAN LYSOZYMES
1B7M 1999-01-24T00:00:00+0000 2.2 VERIFICATION OF SPMP USING MUTANT HUMAN LYSOZYMES

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Lysozyme C P61626 LYSC_HUMAN
100.0 Lysozyme C P61627 LYSC_PANPA
100.0 Lysozyme C P61628 LYSC_PANTR
100.0 Lysozyme C P79179 LYSC_GORGO
99.2 Lysozyme C P79239 LYSC_PONPY
96.9 Lysozyme C P79180 LYSC_HYLLA