Open AccessProduction and purification of human growth‐hormone‐releasing factor from continuous cultures of recombinant‐plasmid‐containing Escherichia coli
Author(s) -
PAGÈS JeanMarie,
BELAICH Anne,
ANBA Jamila,
LAZDUNSKI Claude
Publication year - 1987
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1987.tb13411.x
Subject(s) - recombinant dna , cyanogen bromide , escherichia coli , biochemistry , phosphate , biology , plasmid , microbiology and biotechnology , gene , gene product , chemistry , gene expression , peptide sequence
A recombinant gene comprising phoS (the gene for the phosphate‐binding protein PhoS) fused to a synthetic gene for a modified human growth‐hormone‐releasing factor (mhGRF) has been constructed. This gene was highly expressed in cells growing under conditions of phosphate starvation. Various conditions of continuous culture, varying in phosphate concentrations and dilution rates, have been tested to optimize the expression of the hybrid gene product (PhoS‐mhGRF). Conditions were obtained such that a large amount of the hybrid protein was no longer exported as a result of saturation of export sites, which also induce the inhibition of processing of pre‐PhoE and pre‐OmpA. The pre‐PhoS‐mhGRF, accumulated in the cell, was recovered mainly in the particulate fraction after cell fractionation. This protein was purified. Besides the methionine residues located within the signal sequence, the only other one is located in the fusion joint of the hybrid protein. Thus cyanogen bromide treatment allowed the isolation of pure mhGRF. The yield obtained is about of 1 mg/1 culture.