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Phage display technology involves the surface genetic engineering of phages to expose desirable proteins or peptides whose gene sequences are packaged within phage genomes, thereby rendering direct linkage between genotype with phenotype feasible. This has resulted in phage display systems becoming invaluable components of directed evolutionary biotechnology. The M13 is a DNA phage display system which dominates this technology and usually involves selected proteins or peptides being displayed through surface engineering of its minor coat proteins. The displayed protein or peptide’s functionality is often highly reduced due to harsh treatment of M13 variants. Recently, we developed a novel phage display system using the coliphage Qβ as a nano-biotechnology platform. The coliphage Qβ is an RNA phage belonging to the family of Leviviridae, a long investigated virus. Qβ phages exist as a quasispecies and possess features making them comparatively more suitable and unique for directed evolutionary biotechnology. As a quasispecies, Qβ benefits from the promiscuity of its RNA dependent RNA polymerase replicase, which lacks proofreading activity, and thereby permits rapid variant generation, mutation, and adaptation. The minor coat protein of Qβ is the readthrough protein, A1. It shares the same initiation codon with the major coat protein and is produced each time the ribosome translates the UGA stop codon of the major coat protein with the of misincorporation of tryptophan. This misincorporation occurs at a low level (1/15). Per convention and definition, A1 is the target for display technology, as this minor coat protein does not play a role in initiating the life cycle of Qβ phage like the pIII of M13. The maturation protein A2 of Qβ initiates the life cycle by binding to the pilus of the F+ host bacteria. The extension of the A1 protein with a foreign peptide probe recognizes and binds to the target freely, while the A2 initiates the infection. This avoids any disturbance of the complex and the necessity for acidic elution and neutralization prior to infection. The combined use of both the A1 and A2 proteins of Qβ in this display system allows for novel bio-panning, in vitro maturation, and evolution. Additionally, methods for large library size construction have been improved with our directed evolutionary phage display system. This novel phage display technology allows 12 copies of a specific desired peptide to be displayed on the exterior surface of Qβ in uniform distribution at the corners of the phage icosahedron. Through the recently optimized subtractive bio-panning strategy, fusion probes containing up to 80 amino acids altogether with linkers, can be displayed for target selection. Thus, combined uniqueness of its genome, structure, and proteins make the Qβ phage a desirable suitable innovation applicable in affinity maturation and directed evolutionary biotechnology. The evolutionary adaptability of the Qβ phage display strategy is still in its infancy. However, it has the potential to evolve functional domains of the desirable proteins, glycoproteins, and lipoproteins, rendering them superior to their natural counterparts.

Dystroglycans are essential transmembrane adhesion receptors for laminin. α-Dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of β-dystroglycan. β-Dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin–glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of β-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells.

The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases; the sequences of ovine CPT1A and CPT1B cDNAs have the accession numbers Y18387 and AJ272435 respectively and the partial adipose tissue and liver CPT1A clones have the accession numbers Y18830 and Y18829 respectively. Fatty acid and ketone body metabolism differ considerably between monogastric and ruminant species. The regulation of the key enzymes involved may differ accordingly. Carnitine palmitoyltransferase 1 (CPT 1) is the key locus for the control of long-chain fatty acid beta-oxidation and liver ketogenesis. Previously we showed that CPT 1 kinetics in sheep and rat liver mitochondria differ. We cloned cDNAs for both isoforms [liver- (L-) and muscle- (M-)] of ovine CPT 1 in order to elucidate the structural features of these proteins and their genes ( CPT1A and CPT1B ). Their deduced amino acid sequences show a high degree of conservation compared with orthologues from other mammalian species, with the notable exception of the N-terminus of ovine M-CPT 1. These differences were also present in bovine M-CPT 1, whose N-terminal sequence we determined. In addition, the 5'-end of the sheep CPT1B cDNA suggested a different promoter architecture when compared with previously characterized CPT1B genes. Northern blotting revealed differences in tissue distribution for both CPT1A and CPT1B transcripts compared with other species. In particular, ovine CPT1B mRNA was less tissue restricted, and the predominant transcript in the pancreas was CPT1B. Expression in yeast allowed kinetic characterization of the two native enzymes, and of a chimaera in which the distinctive N-terminal segment of ovine M-CPT 1 was replaced with that from rat M-CPT 1. The ovine N-terminal segment influences the kinetics of the enzyme for both its substrates, such that the K (m) for palmitoyl-CoA is decreased and that for carnitine is increased for the chimaera, relative to the parental ovine M-CPT 1.

Using expressed sequence tag data, we obtained a cDNA for a carnitine palmitoyltransferase I (CPT I)-like molecule from Drosophila melanogaster. The cDNA encodes a 782-residue protein that shows 49% and 48% sequence identity with the rat liver and skeletal-muscle isoforms of CPT I respectively. The sequence has two predicted membrane-spanning regions, suggesting that it adopts the same topology as its mammalian counterparts. The sequence contains all the residues that have been shown to be characteristic of carnitine acetyltransferases. Expression in the yeast Pichia pastoris confirmed that the cDNA does encode a CPT enzyme. The activity was found to be associated with a mitochondria-enriched fraction. Kinetic analysis revealed a K(m) for carnitine of 406 microM and a K(m) for palmitoyl-CoA of 105 microM. The CPT activity was very sensitive to inhibition by malonyl-CoA, with an IC(50) of 0.74 microM when the activity was assayed with 35 microM palmitoyl-CoA and 1% (w/v) albumin at pH 7.0. A histidine residue at position 140 in rat liver CPT I has been indicated to be important for inhibition by malonyl-CoA. The equivalent residue (position 136) in Drosophila CPT I is arginine, implying that any basic residue might be compatible with such sensitivity. Evidence is presented that, unlike in mammals, Drosophila has only a single CPT I gene. Sequences suggesting the existence of a splice variant in the 5' untranslated region were found; this was consistent with the existence of two promoters for the CPT I gene.

The two isoforms of carnitine palmitoyltransferase I (CPT I; muscle (M)- and liver (L)-type) of the mitochondrial outer membrane have distinct kinetic characteristics with respect to their affinity for one of the substrates (l-carnitine) and the inhibitor malonyl-CoA. Moreover, they differ markedly in their hysteretic behavior with respect to malonyl-CoA and in their response to changes in the in vivo metabolic state. However, the two proteins are 62% identical and have the same overall structure. Using liver mitochondria, we have previously shown that the protein is polytopic within the outer membrane, comprising a 46-residue cytosolic N-terminal sequence, two transmembrane segments (TM1 and TM2) separated by a 27-residue loop, and a large catalytic domain (also cytosolic) (Fraser, F., Corstorphine, C. G., and Zammit, V. A. (1997) Biochem. J. 323, 711-718). We have now conducted a systematic study on six chimeric proteins constructed from combinations of three linear segments of rat L- and M-CPT I and on the two parental proteins to elucidate the effects of altered intramolecular interactions on the kinetics of CPT activity. The three segments were (i) the cytosolic N-terminal domain plus TM1, (ii) the loop plus TM2, and (iii) the cytosolic catalytic C-terminal domain. The kinetic properties of the chimeric proteins expressed in Pichia pastoris were studied. We found that alterations in the combinations of the N-terminal plus TM1 and C-terminal domains as well as in the N terminus plus TM1/TM2 pairings resulted in changes in the K(m) values for carnitine and palmitoyl-CoA and the sensitivity to malonyl-CoA of the L-type catalytic domain. The changes in affinity for malonyl-CoA and palmitoyl-CoA occurred independently of changes in the affinity for carnitine. The kinetic characteristics of the M-type catalytic domain and, in particular, its malonyl-CoA sensitivity were much less susceptible to influence by exchange of the other two segments of the protein. The marked difference in the response of the two catalytic domains to changes in the N-terminal domain and TM combinations explains the previously observed differences in the response of L- and M-CPT I to altered physiological state in intact mitochondria and to modulation of altered lipid molecular order of the mitochondrial outer membrane in vivo and in vitro.