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Expression of heparin-binding protein in recombinant mammalian cells

A heparin-binding protein, mammalian technology, applied to mammalian proteins, cells modified by introducing foreign genetic material, animal/human peptides, etc., can solve problems such as HBP heterogeneous populations

Inactive Publication Date: 2002-07-03
LEUKOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In addition, recombinant HBP can be produced in reticulocytes (RBL) (PCT / DK98 / 00275), but heterogeneous populations of HBP with varying degrees of glycosylation are obtained

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0020] Preparation of HBP

[0021] The nucleic acid sequence encoding HBP can be synthesized by well-established standard methods, such as the phosphoamidite method described in S.L.Beaucage and M.H.Caruthers, Tetrahedron Letters 22, 1981, pp1859-1869, or Matthes et al., EMBO J. 3, 1984, the method described in pp801-805. According to the phosphoramidite method, oligonucleotides can be synthesized, such as in an automatic DNA synthesizer, purified, annealed, ligated and cloned into an appropriate vector.

[0022] Techniques for isolating or cloning the nucleic acid sequence of the heparin-binding protein used in the methods of the invention are known in the art, including isolation from genomic DNA, preparation from cDNA, or a combination of both. Cloning of the nucleic acid sequence of the present invention from such genomic DNA can be achieved, for example, by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned...

Embodiment 1

[0041] Embodiment 1: the expression of former HBP in insect cell

[0042] The method used to construct pSX556 is described by Rasmussen et al., 1996, FEBS Lett. 390: 109-112. Prepare the following PCR primers: MHJ 2087: 5′-AAA AAG GAT CCA CCA TGA CCC GGC TGA CAG TCC TGG CCC TGCTGG CTG GTC TGC TGG CGT CCT CGA GGG CCG GCT CCA GCC CCC TTT TGG ACATCG TTG GCG GCC GGA AGG C-3′ ((SEQ ID NO: 15) MHJ 2089: 5'-AAA AAA GCT TCC TAG GCT GGC CCC GGT CCC GGA TTG TTT AAAACG CCA TC-3' (SEQ ID NO: 16)

[0043] MHJ 2087 encodes the BamHI site, the initiation codon and prepro part of the human cDNA (Morgan, J.G. et al., 1991, J. Immun., 147:3210-3214), followed by the first 20 nuclei of the mature part of the gene glycosides.

[0044] MHJ 2089 is complementary to the last 8 codons plus two codons of the coding portion of the HBP gene in the cDNA sequence cited above. It ends with a HindIII site.

[0045] Perform PCR with the following protocol:

[0046] 3 cycles 95°C for 60 seconds, 50°C for...

Embodiment 2

[0051] Example 2: Expression of Δpro-HBP in insect cells

[0052] Prepare an oligopeptide linker (see below) that covers the first 99bp of the HBP sequence (from BamHI to EagI), a fragment that covers the signal peptide and the first 4 amino acids of mature HBP, but does not cover the pre-region (from 73 to EagI). 87), which was replaced by the original BamHI-Eag1 in pSX556 to generate pSX559.

[0053] The linker consists of 4 oligopeptides that anneal in pairs to the following double strands: MHJ2568 / LWN5746: 5'-GATCCACCATGACCCGGCTGACAGTCCTGGCCC-3' (SEQ ID NO: 17) 3'-GTGGTACTGGGCCGACTGTCAGGACCGGGACGACC-5' (SEQ ID NO: 18 ) LWN5745 / MHJ2566: 5'-P-TGCTGGCTGGTCTGCTGGCGTCCTCGAGGGCCATCGTTGGC-3' (SEQ ID NO: 19) 3'-GACCAGACGACCGCAGGAGCTCCCGGTAGCAACCGCCGG-5' (SEQ ID NO: 20)

[0054] SF9 cells were transfected with recombinant baculovirus, and the expressed HBP was purified as described in Example 1. After determining the N-terminal sequence, it was confirmed that from Ile 190% of th...

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Abstract

The present invention relates to a method for producing a mammalian heparin-binding protein in a mammalian cell that can be cultured under anaerobic conditions after introducing a nucleic acid encoding as heparin-binding protein into said cell with comprising (a) introducing into said mammalian cell a nucleic acid encoding said heparin-binding protein; (b) culturing the cell of step (a) under conditions conducive to expression of said HBP; and (c) recovering said HBP from the culture medium. Furthermore, the invention relates to the recombinant mammalian cell used in the method.

Description

field of invention [0001] The present invention relates to a method for producing heparin-binding protein (HBP) in recombinant mammalian cells cultureable under anaerobic conditions after transfection with a nucleic acid encoding a heparin-binding protein. The invention also relates to said recombinant mammalian cells. Background of the invention [0002] The covalent structures of two closely related proteins isolated from peripheral blood neutrophils of human and porcine origin have recently been determined (see H. Flodgaard et al., 1991, Eur. J. Biochem. 197: 535-547; J. Pohl et al., 1990, FEBS Lett. 272:200 et al.). These two proteins have a high degree of similarity to neutrophil elastase, but by selectively incorporating the active serine at position 195 and the active histidine at position 57 (chymotrypsin numbering (B.S.Hartley, 1970, Phil .Trans.Roy.Soc.Series 257:77 ff)) After the mutation, its protease activity was lost. These two protei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00C12N15/09C07K14/47C12N5/10C12N5/22C12P21/02
CPCA61K38/00C07K14/4723C07K14/47
Inventor 汉斯·J·弗洛德加尔德波尔·B·拉斯马森索伦·比约恩伊凡·斯文德森
Owner LEUKOTECH
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