Christer Lindbladh

Selection of phage display combinatorial library peptides with affinity for a yohimbine imprinted methacrylate polymer

α2-Adrenoreceptor mimics, prepared by molecular imprinting of yohimbine, were used to select ligands from a phage display hexapeptide library. Phages with affinity for the yohimbine imprinted methacrylic acid–ethylene glycol dimethacrylate copolymer were selected. Phage affinities were estimated using an enzyme immunoassay. The selected library showed three-fold higher affinity for the imprinted polymer compared with the primary library. Eighty-two of ninety characterized phage clones from the selected library showed low affinity for the polymer. The hexapeptides on eight of these low binding phage clones consisted of mainly hydrophobic amino acid residues, and four clones were identical. The hexapeptides on five of the eight high affinity phage clones contained positively charged amino acids. Identical hexapeptides were expressed on four of these five clones. The results of this study suggest that the majority of the selected phages form hydrophobic and/or ionic interactions with the polymer framework rather than specific interactions only with the yohimbine imprinted sites. Furthermore, a direct correlation can be seen between hexapeptide sequence affinity and the number of positively charged amino acid residues they contain.

Preparation of a genetically fused protein A/luciferase conjugate for use in bioluminescent immunoassays

The genes encoding staphylococcal protein A and bacterial luciferase (Vibrio harveyi) were fused in-frame in order to obtain a general marker enzyme for bioluminescent immunoassays. Two constructs were made where protein A was ligated to the first and the 12th amino acid residue, respectively, of the N terminus of the beta subunit of luciferase. Only the first fusion protein encoding the entire beta subunit was able to form an enzymatically active luciferase complex when expressed together with the alpha subunit. The fusion of protein A to luciferase did not notably alter the emitted wavelength spectrum or its stability to urea treatment. The fusion protein was found to retain at least 50% of the specific bioluminescent activity compared to native luciferase. In preliminary tests, this hybrid protein was shown to be useful in bioluminescent immunoassays.

Recognition in molecularly imprinted polymer α2-adrenoreceptor mimics

Molecularly imprinted polymers (MIPs) selective for the α2-adrenoreceptor antagonist yohimbine (1) have been prepared and studied as α2-adrenoreceptor mimics. Marked ligand stereoselectivity was demonstrated in radioligand binding and HPLC studies upon comparison to blank and corynanthine (2) MIPs. KD values in the nanomolar range have been shown for anti-1 MIP prepared in chloroform solutions upon rebinding in organic media.

Use of genetically prepared enzyme conjugates in enzyme immunoassay

Enzyme immunoassay, using enzymes crosslinked to either antibodies or antigens, has proved a valuable immunological tool for many years. Recently, gene fusion techniques have been used to prepare these enzyme conjugates. This method may be especially advantageous in cases where (1) the antigen is difficult and costly to obtain in large quantities or (2) when the activity of the marker enzyme or the affinity of the antibody or antigen is severely reduced or even destroyed by use of conventional linking methods such as chemical crosslinking. This article uses specific examples to illustrate the potential of gene fusion as a conjugation method.

Selection of a cyclic nonapeptide inhibitor to alpha-chymotrypsin using a phage display peptide library.

A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against α-chymotrypsin (EC 3.4.21.1) at three different pH conditions (6.5, 7.0, and 7.5). Phage peptide clones from the sixth selection, at all three pH conditions, interacted more strongly with α-chymotrypsin than the original library and a wild-type phage did. DNA sequencing of the selected phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions. The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit the enzymatic activity of α-chymotrypsin. The structurally related enzymes trypsin (bovine) and elastase (porcine) were only marginally inhibited by the same peptide under the same conditions. The inhibition constant for α-chymotrypsin was estimated to be 10-6 M. Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified α-chymotrypsin than for natural α-chymotrypsin as determined by an enzyme immunosorbent assay. This peptide phage clone was also competitively prevented from binding to α-chymotrypsin by the corresponding synthetic oxidized peptide. Collectively, the results suggest that the oxidized form of the selected peptide Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of α-chymotrypsin and acts as a specific inhibitor to the enzyme. To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys has not been found in nature.

Selection of peptides with surface affinity for α-chymotrypsin using a phage display library

Peptides with affinity for the surface of alpha-chymotrypsin (EC 3.4.21.1) were selected from a hexapeptide phage display library consisting of approximately 10(7) different clones. Seven selections were performed and five individual phage clones analysed. Compared to the primary library, the five peptide phage clones all interacted more strongly with alpha-chymotrypsin, and DNA sequencing of the phage clones revealed five different amino acid sequences: Gly-Ala-Val-Ile-Thr-His, Arg-Asp-Ile-Val-Val-Ala, Val-Tyr-Ser-His-Ala-Ser, Gly-Ser-Tyr-Ser-Ala-Gly and Leu-Asp-Ile-Val-Val-Ala. Two of the peptides exhibited 83% identity (i.e. a difference of just one amino acid). The chemically synthesized peptides competitively reduced the binding of the corresponding peptide phage clone to alpha-chymotrypsin. Binding of some of the selected peptide phage clones to alpha-chymotrypsin was also reduced by several of the other non-corresponding synthesized peptides, suggesting that these peptides have common recognition areas on the enzyme. Three of the synthesized peptides were poor substrates of alpha-chymotrypsin and they did not inhibit enzyme activity. Our results suggest that it is possible to select peptides from peptide phage display libraries with affinity for different surface structures on the enzyme, not involved in the biologically active site.

Standard calibration proteins for Western blotting obtained by genetically prepared protein A conjugates

Genetically prepared protein A fusion proteins, having retained antibody binding capacity, were used to design different well-defined standard molecular weight marker proteins for Western blotting. The blotted marker proteins are developed at the same time and with the same reagents as the protein sample of interest.

Enzymatic coupling of two d-amino acid residues in aqueous media

Abstract The formation of a D-Ala-D-Ala containing tripeptide in aqueous solution, catalysed by muramoylpentapeptide carboxypeptidase (E.C. 3.4.17.8), is described.

“Togetherness” Between Proteins Generated By Gene Fusion

Several artificial bi- and polyfunctional enzymes have been prepared by gene fusion in vitro . These recombinant enzymes catalyze consecutive reactions and often exhibit favorable proximity effects due to the closeness of the active sites. This is indicated by channeling of the intermediate substrate and reduced lag phases. Applications can be found in enzymatic analysis and metabolic engineering. The potential of gene fusion as a conjugation method in enzyme immunoassay is also discussed.

Structure of a cyclic peptide with a catalytic triad, determined by computer simulation and NMR spectroscopy.

SummaryWe report the design of a cyclic, eight-residue peptide that possesses the catalytic triad residues of the serine proteases. A manually built model has been relaxed by 0.3 ns of molecular dynamics simulation at room temperature, during which no major changes occurred in the peptide. The molecule has been synthesised and purified. Two-dimensional NMR spectroscopy provided 35 distance and 7 torsion angle constraints, which were used to determine the three-dimensional structure. The experimental conformation agrees with the predicted one at the β-turn, but deviates in the arrangement of the disulphide bridge that closes the backbone to a ring. A 1.2 ns simulation at 600 K provided extended sampling of conformation space. The disulphide bridge reoriented into the experimental arrangement, producing a minimum backbone rmsd from the experimental conformation of 0.8 Å. At a later stage in the simulation, a transition at Ser3 produced more pronounced high-temperature behaviour. The peptide hydrolyses p-nitrophenyl acetate about nine times faster than free histidine.