Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector.
In order to express human serum retinol-binding protein (sRBP) in Escherichia coli in a form that is structurally indistinguishable from the native protein, we placed the coding sequence of the RBP cDNA next to that of the outer membrane protein A (OmpA) signal sequence in the secretion vector, pIN-III-OmpA1. However, this construct did not generate detectable expression of RBP in E. coli. When the DNA fragment consisting of the ribosome-binding site and the OmpA-RBP fusion sequence was subcloned downstream to the T7 promoter of pKS-Bluescript, however, the resultant construct (pOmp-RBP2) gave low but detectable secretion of RBP into the periplasm. Deletion of the 3' untranslated region of the RBP cDNA (pOmp-RBP3) further improved the expression (by approx. 20-fold). After charging with retinol, the secreted RBP was purified from the periplasm on a transthyretin-affinity resin. The purified protein exhibited all the three molecular recognition properties characteristic of sRBP, i.e. it interacted with retinol, transthyretin and its cell-surface receptor. Comparison of the receptor binding properties of the recombinant RBP (rRBP) with those of the serum protein revealed that while the affinity of rRBP is similar to sRBP (50 +/- 20 nM), the Bmax of the rRBP is about 6-8-fold higher. This indicates that a major proportion of RBP, isolated from serum, is incapable of interacting with the receptor.
Sivaprasadarao
A
A
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Findlay
J B
JB
D001345:Autoradiography; D001483:Base Sequence; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D010920:Placenta / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012177:Retinol-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013462:Sulfur Radioisotopes
D017931:DNA Primers; D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine