High-level expression of human TFF3 in Escherichia coli.
ABSTRACT A strategy for expression and purification of recombinant N-terminal human trefoil factor family-domain peptide 3 (hTFF3) in Escherichia coli was established. The gene of hTFF3 was synthesized to substitute the low-usage condons with corresponding high-usage synonymous condons. At the same time, the signal peptide of DsbC was added to the N-terminus of the hTFF3 gene. The mature recombinant hTFF3 was located in the periplasm of E. coli, which can be released by sonication. The protein was further purified by a two-step cation exchange chromatography mentod. The yield is about 14-15 mg/l of culture. The biological activity of purified hTFF3 was analyzed by cell-based apoptosis assay, which shows that the recombinant hTFF3 is biologically active.
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ABSTRACT: Porcine pancreatic spasmolytic polypeptide (PSP) belongs to a large family of homologous growth factor-like polypeptides characterized by a disulfide-linked "trefoil motif," duplicated and conserved in various family members. PSP contains two trefoil motifs, has several pharmacological actions on the gut, and has growth factor properties on epithelial cells in vitro. The human PSP analogue, human spasmolytic polypeptide, appears to be involved in many regenerative situations and, especially, in healing gastrointestinal ulcers. One member of the trefoil family, pS2, is secreted in ≈50% of estrogen-dependent human breast carcinomas, which has led to its use as a tumor prognostic marker. Both pS2 and human spasmolytic polypeptide are also widely expressed in chronic gastrointestinal ulcerative conditions such as Crohn disease. Here we report the three-dimensional structure at 2.6-Å resolution of a trefoil-containing protein, namely PSP, purified from porcine pancreas. The structure shows two homologous domains that share a supersecondary structure and disulfide bond pattern. The two domains pack asymmetrically giving rise to a number of protruding loops, exposed clefts, and an unusual electrostatic surface potential. Knowledge of the structure of PSP should allow the design of mutants to investigate further the function of PSP and other trefoil-containing peptides.Proceedings of The National Academy of Sciences - PNAS. 01/1994; 91(3):1084-1088.
- Genomics 03/1997; 40(1):200-2. · 3.01 Impact Factor
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ABSTRACT: Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme. In the presence of Mg2+, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg2+ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg2+. Mg2+ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose. These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.Biochimica et Biophysica Acta 10/1976; 443(3):534-44. · 4.66 Impact Factor