Bovine Heart Fatty Acid-binding Protein Research Articles

The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins

We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag 15 is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the Nano-tag 9 is a 9-mer peptide with a dissociation constant of 17 nM. We demonstrate the one-step purification of Nano-tagged proteins, namely bovine heart fatty acid-binding protein (FABP), bacterial chloramphenicol acetyltransferase (CAT), and green fluorescent protein (GFP), from an in vitro translation system as well as from an Escherichia coli lysate. No significant influence of the Nano-tag 15 and of the conditions during affinity chromatography on maturation or activity of the proteins was observed whereas the Nano-tag 9 revealed a slight decline in the amount and activity of the synthesized proteins. The main advantage of the Nano-tag is the mild and specific elution with washing buffer plus biotin or related compounds, which enables the elution of the bound fusion protein from the streptavidin column in the native state. Additionally, the Nano-tag allowed the detection of recombinant proteins on Western blots by a streptavidin–alkaline phosphatase conjugate.

Searching Sequence Space for High-affinity Binding Peptides using Ribosome Display

We present the construction of a synthetic library based on the scaffold of bovine heart fatty acid-binding protein (FABP) with 1.1×10 14 independent members. Ribosome display was applied to select streptavidin-binding peptides in vitro from 2×10 13 molecules of the library each encoding FABP with 15 contiguous random amino acid residues at its N terminus. The selection yielded several different binding peptides. The best binder possessed a dissociation constant as low as 4 nM and, in contrast to the previously isolated peptides, contained no HPQ motif. A substitution analysis enabled shortening of the 15-mer peptide and revealed a 9-mer variant with a dissociation constant of 17 nM, which is a 1000-fold increase of affinity compared to the already known peptides of this size. This high-affinity binding peptide in combination with the whole set of streptavidin conjugates should be an extremely useful tool for the detection and purification of recombinant proteins.

An improved protein bioreactor: efficient product isolation during in vitro protein biosynthesis via affinity tag.

In vitro protein biosynthesis became a powerful technology for biochemical research. Beside the determination of structure and function in vitro selection of proteins is also of great interest. In most cases the use of a synthesized protein for further applications depends on its purity. For this purpose the in vitro production and purification of proteins with short affinity tails was established. A cell-free protein synthesis system was employed to produce bovine heart fatty acid-binding protein and bacterial chloramphenicol acetyltransferase with and without fusion of the Strep-tag affinity peptide. The quantitative removal of fusion protein during cell-free synthesis from a batch reaction and a semicontinuous flow cell-free reactor were achieved. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the proteins were observed. The product removal from the continuous flow cell-free reactor is still an only partially solved problem, because the use of ultrafiltration membranes has some limitations. The results document that it should be possible to avoid these limitations by introducing an affinity system.

Presence of two new fatty acid binding proteins in catfish liver.

A basic fatty acid binding protein (FABP), closely related to that of chicken liver, was isolated and characterized from catfish (Rhamdia sapo) liver in a previous work. Results herein show the presence of another two FABPs in which partial amino acid sequences reveal great similarity with the corresponding sequences of other already known FABPs belonging to the heart type. The purification procedures for both proteins involve gel filtration, anion-exchange chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (as a last step). Because both FABP N-termini were blocked, they were submitted to in-gel tryptic digestion and the resulting peptides were separated by high performance liquid chromatography, and sequenced by Edman degradation. One of these proteins presented the highest identity percentage when compared with those of the human and bovine heart and bovine brain (81%), and the other when compared with those of chicken retina (75%) and mouse and bovine heart FABP (70%). The presence of several FABPs plus the fact that they belong to different types, as found in the Rhamdia sapo liver, is unusual in mammals, which express a characteristic liver-type member of this protein family.

Mammary Derived Growth Inhibitor Is Not a Distinct Protein but a Mix of Heart-type and Adipocyte-type Fatty Acid-binding Protein

The amino acid sequence of the mammary derived growth inhibitor (MDGI) from bovine mammary gland (Böhmer, F.-D., Kraft, R., Otto, A. , Wernstedt, C., Hellman, U., Kurtz, A., Müller, T., Rohde, K., Etzold, G., Lehmann, W., Langen, P., Heldin, C.-H., and Grosse, R. (1987) J. Biol. Chem. 262, 15137-15143) revealed 95% identity to bovine heart fatty acid-binding protein (H-FABP), explaining the observed immunocross-reactivity. However, a cDNA encoding MDGI has not been found to date. Artificial MDGI cDNA was expressed in an in vitro transcription/translation assay. Analysis by isoelectric focusing of the immunoprecipitated in vitro translation products of lactating bovine mammary gland mRNA did not indicate a protein corresponding to the in vitro translation product of artificial MDGI mRNA. Moreover, two-dimensional electrophoresis of bovine mammary gland proteins confirmed the absence of a protein with the pI of the in vitro translated artificial MDGI mRNA in bovine mammary gland and instead revealed, apart from H-FABP, an unknown protein that was recognized by anti-H-FABP antibodies. From lactating bovine mammary gland the cDNA for adipocyte fatty acid-binding protein (A-FABP) was cloned. The in vitro translation of recombinant mRNA derived from this cDNA yielded a polypeptide that behaved like the unknown immunoreactive protein. Western blotting and immunofluorescence using monospecific antibodies demonstrated the coexistence of H-FABP and A-FABP in the lactating mammary gland. Taking into account that deviations of the MDGI sequence from the bovine H-FABP sequence correspond with A-FABP we attribute the structure originally reported as MDGI to a mix of these proteins.

Enzyme immunosensor for diagnosis of myocardial infarction

Amperometric enzyme immunosensors have been developed for the detection of the early myocardial infarction marker heart fatty acid binding protein (H-FABP) in blood plasma. They are based on anti-H-FABP antibodies immobilized on nitrocellulose or activated nylon membranes covering a modified Clark-type oxygen electrode. Two approaches have been tested using bovine H-FABP as a model: (i) the competition principle, where defined amounts of glucose oxidase (GOD)-labelled H-FABP compete with free H-FABP for binding to the immobilized capture antibodies; (ii) the sandwich principle, where the free H-FABP binds to the immobilized capture antibody and to a secondary GOD-labelled antibody, forming a sandwich. In both approaches, after addition of glucose, the activity of the enzyme label is determined with the amperometric electrode within minutes. The immunosensor using the sandwich principle on activated nylon membranes can be used repeatedly for at least 10 measurements after regeneration of the antibody membranes without loss of binding capacity of the capture antibodies. For the determination of human H-FABP, the sandwich principle with monoclonal anti-bovine-H-FABP antibodies as capture and GOD-labelled goat-anti-human-H-FABP antibodies as secondary antibodies is chosen. Initial experiments demonstrate that the sensitivity of the immunosensor is sufficient for the determination of clinical levels of H-FABP in plasma after myocardial infarction (100–300 ng ml −1).

Studies on the effect of an heterologous fatty acid-binding protein on acyl-CoA oxidase induction in Saccharomyces cerevisiae.

The participation of fatty acid-binding protein (FABP) in the induction of peroxisomal beta-oxidation of fatty acids was investigated in vivo in an heterologous system. Bovine heart FABP was expressed in Saccharomyces cerevisiae under the control of two different promoters: a constitutive one and an oleic acid-inducible one. Constructs were introduced into yeast cells on multicopy and integrating plasmids. The heterologous FABP was present in yeast cells in two isoforms having pI values of about 5 and was able to bind oleic acid. The heterologous FABP had no significant effect on acyl-CoA oxidase activity at various concentrations of the inducing agent.

N-terminal variants of fatty acid-binding protein from bovine heart overexpressed in Escherichia coli

An expression vector for bovine heart fatty acid-binding protein (H-FABP) was constructed by introducing the coding part of the cDNA into the pET-3d vector. Transformed Escherichia coli strain BL21 (DE3)pLysS produced functional recombinant H-FABP up to 40% of the soluble proteins. The expression of fatty acid-binding protein was under the control of the T7-phi 10 promoter and the corresponding T7-RNA-polymerase in turn was induced by isopropyl β- d-thiogalactopyranoside. By combination of cation exchange chromatography and gel filtration pure recombinant protein was obtained exhibiting isoelectric heterogeneity. Recombinant H-FABP was resolved into at least six variants with isoelectric points between 5.1 and 5.6. After separation by preparative isoelectric focusing the four major variants were digested with trypsin and the resulting peptides were characterized by high performance liquid chromatography (HPLC), matrix assisted laser desorption/ionization (MALDI) mass spectrometry, amino acid sequencing and chemical modification. The structural differences were traced back to the N-termini beginning with either methionine, as expected from the cDNA, or methionine sulfoxide, valine and N-formyl methionine. The latter three arise from oxidation, cleavage of N-terminal methionine and incomplete deformylation, respectively.

Solution structure of bovine heart fatty acid-binding protein (H-FABPc).

Fatty acid-binding protein (FABP) from bovine heart, a 15 kDa cytoplasmic protein has been investigated by multi-dimensional homonuclear and heteronuclear NMR-spectroscopy. Perdeuterated palmitic acid has been used as fatty acid ligand. The tertiary structure has been determined from distance geometry calculations with the variable target functions algorithm (DIANA) utilizing 1027 interproton distance constraints, which were obtained from 1H-homonuclear NOESY spectra. Overlapping NOE crosspeaks were assigned by heteronuclear multidimensional NMR-experiments with a 15N-labelled sample. The tertiary structure resembles a beta-barrel (beta-clam) consisting of ten anti-parallel beta-strands and a short helix-turn-helix motif. The beta-strands are arranged in two nearly orthogonal beta-sheets composed of 5 strands each. The solution structure is compared with the x-ray crystal structure of bovine heart and rat intestinal FABPs.

Cardiac fatty acid-binding proteins. Isolation and characterization of the mitochondrial fatty acid-binding protein and its structural relationship with the cytosolic isoforms.

In the course of our studies on the structural diversity of the isoforms of cardiac fatty acid-binding proteins (cFABPs), a cardiac-type FABP from the matrix of bovine heart mitochondria was purified to homogeneity and obtained as a single 15-kDa protein with an isoelectric point of 4.9. The primary structures of this protein and of the two isoforms isolated from the cytosol (pI4.9-cFABP and pI 5.1-cFABP) were investigated by means of plasma desorption mass spectrometry and sequencing of peptides. All three proteins are amino-terminally blocked with an acetyl group and shown to be colinear with the sequence deduced from a cDNA clone for bovine heart fatty acid-binding protein (Billich, S., Wissel, T., Kratzin, H., Hahn, U., Hagenhoff, B., Lezius, A. G., and Spener, F. (1988) Eur. J. Biochem. 175, 549-556) except for the residue at position 98. This residue is demonstrated to be the molecular origin of bovine cFABP isoforms since pI 5.1-cFABP contains Asn98 in accordance with the sequence derived from the cDNA, whereas in pI 4.9-cFABP, this position is occupied by Asp98. Moreover, mitochondrial FABP is identical to pI 4.9-cFABP. Molecular masses of pI 4.9-cFABP (14,679 +/- 10 Da) and pI 5.1-cFABP (14,678 +/- 20 Da) determined by plasma desorption mass spectrometry coincide with that calculated from the cDNA (14,673 Da). Hence, residues linked to these proteins by posttranslational modification are not present, and the Asn-Asp exchange is the sole origin of heterogeneity of mitochondrial and cytosolic fatty acid-binding proteins from bovine heart.

Expression of a functionally active cardiac fatty acid-binding protein in the yeast, Saccharomyces cerevisiae

The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size to native protein as judged from SDS-polyacrylamide gel electrophoresis, was reached after approximately 16 hours of induction. Analysis of particulate and soluble subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind 14C-oleic acid in an in vitro assay. Growth of the transformants on galactose as the carbon source was significantly retarded at 37 degrees C. Whereas the fatty acid pattern of total lipids was not altered in transformed cells, desaturation of exogenously added 14C-palmitic acid was significantly reduced both at 30 and 37 degrees C. The lowest percentage of radioactively labeled unsaturated fatty acids was found in the phospholipid fraction.

Cloning of a full-length complementary DNA for fatty-acid-binding protein from bovine heart.

A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lambda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by Böhmer et al. [J. Biol. Chem. 262, 15137-15143 (1987)]. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.