Characterization and In Silico Analysis of The Structural Features of G-CSF Derived from Lysates of Escherichia coli

Abstract

Objective: Granulocyte colony-stimulating factor (G-CSF) has a wide variety of functions including stimulation of hematopoiesis and proliferation of granulocyte progenitor cells. Recombinant human G-CSF (rh-G-CSF) is used for treatment of neutropenia in patients receiving chemotherapy. The mature bloodstream neutrophils express G-CSF receptor (G-CSFR), presenting a significant and specific mechanism for circulating G-CSF clearance. Computational studies are essential bioinformatics methods used for characterization of proteins with regard to their physicochemical properties and 3D configuration, as well as protein-ligand interactions for recombinant drugs. We formerly produced rh-G-CSF in E. coli and showed that the isolated protein had unacceptable biological activity in mice. In the present paper, we aimed to characterize the purified rh-G-CSF by analytical tests and developed an in vivo model by computational modelling of G-CSF.

Materials and methods: In this experimental study, we analyzed the purified G-CSF using the analytical experiments. Then, the crystalline structure was extracted from Protein Data Bank (PDB) and molecular dynamics (MD) simulation was performed using Gromacs 5.1 package under an Amber force field. The importance of amino acid contents of G-CSF, to bind the respective receptor was also detected; moreover, the effect of dithiothreitol (DTT) used in G-CSF purification was studied.

Results: The results revealed that characteristics of the produced recombinant G-CSF were comparable with those of the standard G-CSF and the recombinant G-CSF with the residual amino acid was stable. Also, purification conditions (DTT and existence of extra cysteine) had a significant effect on the stability and functionality of the produced G-CSF.

Conclusion: Experimental and in silico analyses provided good information regarding the function and characteristics of our recombinant G-CSF which could be useful for industrial researches.

Keywords: Characterization; E. coli; Granulocyte Colony-Stimulating Factor.

Fig.1

Overall schema of 2800 bp expression vector with g-csf gene and 2 different inteins genes, pelB signal sequence and intervening sequence. A. Schematic representation of the vector construction and the expression of the N-terminal fusion and C-terminal fusion proteins and B. In C-terminal fusion protein, intein2-CBD was fused to the C-terminus of h G-CSF, allowing the cleavage of h G-CSF from G-CSF-intein2 using DTT. In N-terminal fusion protein, CBD-intein1 was fused to the N-terminus of h G-CSF, allowing the cleavage of h G-CSF from intein1- G-CSF through a pH and temperature shift. G-CSF; Granulocyte colony-stimulating factor.

Fig.2

Isoelectric focusing of purified rh G-CSF and PDgrastim (standard) in the strips of pH=3-10. Both of the standard and purified proteins were stopped in pH=6.1 and no impurity or no band was found. G-CSF; Granulocyte colony-stimulating factor.

Fig.3

The reversed phase HPLC chromatogram for the Endopeptidase digest of purified rh G-CSF in comparison with standard rh G-CSF. A. Peptide map chromatograms of PDgrastim and B. Purified rh G-CSF. G-CSF; Granulocyte colony-stimulating factor.

Fig.4

Results of molecular dynamic simulation of RMSF and RMSD diagram. A. Comparison of RMSD changes. The simulation of G-CSF protein in the presence and absence of DTT, B. Comparison of the RMSF diagram of G-CSF in the presence and absence of DTT, C. RMSF diagram of GCSF protein in complex with GCSFR in the presence of DTT, D. The RMSD variations associated with the natural GCSF protein and GCSF have Cysteine amino acid, and E. RMSF diagram shows flexible amount of single amino acids for normal and atypical protein (contains cysteine). RMSF; Root-mean-square fluctuation, RMSD; Root-mean-square deviation, G-CSF; Granulocyte colony stimulating factor, and DTT; Dithiothreitol.