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.
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页码:209 / 215
页数:7
相关论文
共 24 条
  • [1] A MOLECULAR-MODEL FOR THE RETINOL BINDING-PROTEIN TRANSTHYRETIN COMPLEX
    AQVIST, J
    TAPIA, O
    [J]. JOURNAL OF MOLECULAR GRAPHICS, 1992, 10 (02): : 120 - 123
  • [2] BAVIK CO, 1992, J BIOL CHEM, V267, P23035
  • [3] STRUCTURE OF PRE-ALBUMIN - SECONDARY, TERTIARY AND QUATERNARY INTERACTIONS DETERMINED BY FOURIER REFINEMENT AT 1.8-A
    BLAKE, CCF
    GEISOW, MJ
    OATLEY, SJ
    RERAT, B
    RERAT, C
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1978, 121 (03) : 339 - 356
  • [4] COLONTUONI V, 1983, NUCLEIC ACIDS RES, V11, P7769
  • [5] CRYSTALLOGRAPHIC REFINEMENT OF HUMAN SERUM RETINOL BINDING-PROTEIN AT 2A RESOLUTION
    COWAN, SW
    NEWCOMER, ME
    JONES, TA
    [J]. PROTEINS-STRUCTURE FUNCTION AND GENETICS, 1990, 8 (01): : 44 - 61
  • [6] COMPARISON OF THE UPTAKE AND METABOLISM OF RETINOL DELIVERED TO PRIMARY MOUSE KERATINOCYTES EITHER FREE OR BOUND TO RAT SERUM RETINOL-BINDING PROTEIN
    CREEK, KE
    SILVERMANJONES, CS
    DE LUCA, LM
    [J]. JOURNAL OF INVESTIGATIVE DERMATOLOGY, 1989, 92 (02) : 283 - 289
  • [7] SECRETION CLONING VECTORS IN ESCHERICHIA-COLI
    GHRAYEB, J
    KIMURA, H
    TAKAHARA, M
    HSIUNG, H
    MASUI, Y
    INOUYE, M
    [J]. EMBO JOURNAL, 1984, 3 (10) : 2437 - 2442
  • [8] Goodman DS, 1984, RETINOIDS, V2, P42
  • [9] SECRETION OF BETA-LACTAMASE REQUIRES THE CARBOXY END OF THE PROTEIN
    KOSHLAND, D
    BOTSTEIN, D
    [J]. CELL, 1980, 20 (03) : 749 - 760
  • [10] MELHUS H, 1992, J BIOL CHEM, V267, P12036