EXPRESSION OF FUNCTIONAL HUMAN RETINOL-BINDING PROTEIN IN ESCHERICHIA-COLI USING A SECRETION VECTOR

被引:17
作者
SIVAPRASADARAO, A
FINDLAY, JBC
机构
[1] Dept Biochemistry/Molecular Biology, University of Leeds
关键词
D O I
10.1042/bj2960209
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-OmpAl. 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 B(max) 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.
引用
收藏
页码:209 / 215
页数:7
相关论文
共 24 条
  • [11] RETINOL-BINDING PROTEIN AND TRANSTHYRETIN EXPRESSED IN HELA-CELLS FORM A COMPLEX IN THE ENDOPLASMIC-RETICULUM IN BOTH THE ABSENCE AND THE PRESENCE OF RETINOL
    MELHUS, H
    NILSSON, T
    PETERSON, PA
    RASK, L
    [J]. EXPERIMENTAL CELL RESEARCH, 1991, 197 (01) : 119 - 124
  • [12] 3-DIMENSIONAL STRUCTURE AND ACTIVE-SITE OF 3 HYDROPHOBIC MOLECULE-BINDING PROTEINS WITH SIGNIFICANT AMINO-ACID-SEQUENCE SIMILARITY
    MONACO, HL
    ZANOTTI, G
    [J]. BIOPOLYMERS, 1992, 32 (04) : 457 - 465
  • [13] THE 3-DIMENSIONAL STRUCTURE OF RETINOL-BINDING PROTEIN
    NEWCOMER, ME
    JONES, TA
    AQVIST, J
    SUNDELIN, J
    ERIKSSON, U
    RASK, L
    PETERSON, PA
    [J]. EMBO JOURNAL, 1984, 3 (07) : 1451 - 1454
  • [14] PETERSON PA, 1971, J BIOL CHEM, V246, P34
  • [15] VITAMIN-A SUPPLY OF THE CORNEA
    RASK, L
    GEIJER, C
    BILL, A
    PETERSON, PA
    [J]. EXPERIMENTAL EYE RESEARCH, 1980, 31 (02) : 201 - 211
  • [16] STRUCTURE-FUNCTION STUDIES OF MURINE EPIDERMAL GROWTH-FACTOR - EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF EPIDERMAL GROWTH-FACTOR GENE
    RAY, P
    MOY, FJ
    MONTELIONE, GT
    LIU, JF
    NARANG, SA
    SCHERAGA, HA
    WU, R
    [J]. BIOCHEMISTRY, 1988, 27 (19) : 7289 - 7295
  • [17] RAZ A, 1970, J BIOL CHEM, V245, P1903
  • [18] Sambrook J., 1989, MOL CLONING LAB MANU
  • [19] CHARACTERISTICS OF RETINOL ACCUMULATION FROM SERUM RETINOL-BINDING PROTEIN BY CULTURED SERTOLI CELLS
    SHINGLETON, JL
    SKINNER, MK
    ONG, DE
    [J]. BIOCHEMISTRY, 1989, 28 (25) : 9641 - 9647
  • [20] SIVAPRASADARAO A, 1988, BIOCHEM J, V255, P561