TY - JOUR
T1 - A strategy for bacterial production of a soluble functional human neonatal Fc receptor
AU - Andersen, Jan Terje
AU - Justesen, Sune
AU - Berntzen, Gøril
AU - Michaelsen, Terje E
AU - Lauvrak, Vigdis
AU - Fleckenstein, Burkhard
AU - Buus, Søren
AU - Sandlie, Inger
N1 - Keywords: Albumins; Bioreactors; Circular Dichroism; Cloning, Molecular; Escherichia coli; Genetic Vectors; Histocompatibility Antigens Class I; Humans; Immunoglobulin G; Protein Folding; Receptors, Fc; Recombinant Proteins; beta 2-Microglobulin
PY - 2008
Y1 - 2008
N2 - The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus (human) or newborn (rodents), and may translocate IgG over mucosal surfaces. FcRn interacts with the Fc-region of IgG and domain III of albumin with binding at pH 6.0 and release at pH 7.4. Knowledge of these interactions has facilitated design of recombinant proteins with altered serum half-lives and/or altered biodistribution. To generate further research in this field, there is a great need for large amounts of soluble human FcRn (shFcRn) for in vitro interaction studies. In this report, we describe a novel laboratory scale production of functional shFcRn in Escherichia coli (E. coli) at milligram level. Truncated wild type hFcRn heavy chains were expressed, extracted, purified from inclusion bodies under denaturing non-reducing conditions, and subsequently refolded in the presence of human beta(2)-microglobulin (hbeta(2)m). The secondary structural elements of refolded heterodimeric shFcRn were correctly formed as demonstrated by circular dichroism (CD). Furthermore, functional and stringent pH dependent binding to IgG and human serum albumin were demonstrated by ELISA and surface plasmon resonance (SPR). This method may be easily adapted for the expression of large amounts of other FcRn species and MHC class I related molecules.
AB - The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus (human) or newborn (rodents), and may translocate IgG over mucosal surfaces. FcRn interacts with the Fc-region of IgG and domain III of albumin with binding at pH 6.0 and release at pH 7.4. Knowledge of these interactions has facilitated design of recombinant proteins with altered serum half-lives and/or altered biodistribution. To generate further research in this field, there is a great need for large amounts of soluble human FcRn (shFcRn) for in vitro interaction studies. In this report, we describe a novel laboratory scale production of functional shFcRn in Escherichia coli (E. coli) at milligram level. Truncated wild type hFcRn heavy chains were expressed, extracted, purified from inclusion bodies under denaturing non-reducing conditions, and subsequently refolded in the presence of human beta(2)-microglobulin (hbeta(2)m). The secondary structural elements of refolded heterodimeric shFcRn were correctly formed as demonstrated by circular dichroism (CD). Furthermore, functional and stringent pH dependent binding to IgG and human serum albumin were demonstrated by ELISA and surface plasmon resonance (SPR). This method may be easily adapted for the expression of large amounts of other FcRn species and MHC class I related molecules.
U2 - 10.1016/j.jim.2007.11.003
DO - 10.1016/j.jim.2007.11.003
M3 - Journal article
C2 - 18155020
SN - 0022-1759
VL - 331
SP - 39
EP - 49
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -