Lpl-gpihbp1 fusion polypeptides
A technology for fusion of peptides and peptides, applied in the direction of fusion peptides, peptides containing His tags, peptides containing affinity tags, etc., can solve problems such as expensive and difficult to achieve HTAP prevention
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[0174] general method
[0175] expression plasmid
[0176] Mammalian expression vectors of LPL, GPIHBP1, ANGPTL3 and ANGPTL4 were synthesized, and the sequences of the open reading frames are listed in Table 2.
[0177] Table 2 Amino acid sequences of recombinant human LPL, soluble human GPIHBP1, human ANGPTL3, human ANGPTL4 and human LPL-GPIHBP1 fusion polypeptide
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[0181] Enzymes and Reagents
[0182] Amplex Red, resorufin butyrate, Enzchek and DGGR substrates were purchased from Life Technologies. Human VLDL and chylomicrons were from EMD Millipore and Athens research, respectively. BSA was obtained from Sigma. HR series NEFA-HR(2) chromogen A and HR series NEFA-HR(2) chromogen B were purchased from Wako Diagnostics. Detergents were obtained from Sigma Corporation.
[0183] Expression and purification of recombinant proteins
[0184] Human LPL: Recombinant human LPL was prepared using the following procedure. HEK293T cells...
example 1
[0215] Example 1: GPIHBP1 stabilizes LPL, prevents its aggregation, and increases lipase activity
[0216] Initial attempts were made to purify the LPL protein by itself. To facilitate this purification, various LPL constructs were synthesized either untagged or with N-terminal or C-terminal tags. These LPL constructs were expressed in mammalian cells and purified using heparin chromatography or Ni affinity chromatography. The purified protein was found to be active but highly aggregated ( figure 1 , panels A, C and D). Co-transfection of LPL with LMF1 did not improve the yield of LPL, and the purified protein remained highly aggregated (data not shown). Coexpression of a purified soluble form of GPIHBPI with LPL protected LPL from spontaneous inactivation (data not shown). Then, GPIHBP1 was co-expressed with LPL in the presence of LMF1 in the following manner: N-terminal 6-His-tagged LPL, untagged GPIHBP1 and LMF1 were co-transfected into HEK293T cells at a ratio of 3:1...
example 2
[0217] Example 2: LPL-GPIHBP1 fusion polypeptide is homogeneous, stable, and has high specific activity
[0218] A non-dissociated complex of LPL and GPIHBP1 was then generated by making an LPL-GPIHBP1 fusion construct. Mammalian expression vectors were designed with LPL and soluble GPIHBP1 open reading frames linked via a 20 amino acid serine / glycine linker. To aid in purification, a purification tag was added to the N- or C-terminus of the fusion construct. figure 2 Panel A shows FLAG-6-His-AviTag with C-terminus TM (FHA)-tagged purified LPL-GPIHBP1 complex. Fusion polypeptides greater than 95% pure were obtained using Ni affinity chromatography and size exclusion chromatography ( figure 2 , Panel A). Similar to the LPL / GPIHBP1 coexpressed complex, the resulting fusion polypeptide was free of aggregates and resolved into a single homogeneous species with a molecular weight of approximately 75 KDa by size exclusion chromatography ( figure 2 , panel B). These data i...
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