A strategy for bacterial production of a soluble functional human neonatal Fc receptor

Jan Terje Andersen; Sune Justesen; Gøril Berntzen; Terje E Michaelsen; Vigdis Lauvrak; Burkhard Fleckenstein; Søren Buus; Inger Sandlie

doi:10.1016/j.jim.2007.11.003

A strategy for bacterial production of a soluble functional human neonatal Fc receptor

Jan Terje Andersen, Sune Justesen, Gøril Berntzen, Terje E Michaelsen, Vigdis Lauvrak, Burkhard Fleckenstein, Søren Buus, Inger Sandlie

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

25 Citationer (Scopus)

Abstract

Udgivelsesdato: 2008-Feb-29

Originalsprog	Engelsk
Tidsskrift	Journal of Immunological Methods
Vol/bind	331
Udgave nummer	1-2
Sider (fra-til)	39-49
Antal sider	10
ISSN	0022-1759
DOI	https://doi.org/10.1016/j.jim.2007.11.003
Status	Udgivet - 2008

Bibliografisk note

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

Adgang til dokumentet

10.1016/j.jim.2007.11.003

Citationsformater

A strategy for bacterial production of a soluble functional human neonatal Fc receptor. / Andersen, Jan Terje; Justesen, Sune; Berntzen, Gøril; Michaelsen, Terje E; Lauvrak, Vigdis; Fleckenstein, Burkhard; Buus, Søren; Sandlie, Inger.

I: Journal of Immunological Methods, Bind 331, Nr. 1-2, 2008, s. 39-49.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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title = "A strategy for bacterial production of a soluble functional human neonatal Fc receptor",

abstract = "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.",

author = "Andersen, {Jan Terje} and Sune Justesen and G{\o}ril Berntzen and Michaelsen, {Terje E} and Vigdis Lauvrak and Burkhard Fleckenstein and S{\o}ren Buus and Inger Sandlie",

note = "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",

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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

VL - 331

SP - 39

EP - 49

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 1-2

ER -