A new protein conjugate that replaces the use of secondary antibodies engineered from the two staphylococcal enzymes protein A and 6-phospho-{beta}-galactosidase

The lacG gene encoding the 6-phospho-ß-galactosidase(E.C.3.2.1.85) of Staphylococcus aureus was fused to the proteinA gene in the plasmid pRIT2T. Escherichia coli cells containingthis plasmid produce a fusion protein with both IgG bindingand 6-phospho-ß-galactosidase activities after heatinduction. The recombinant gene was overexpressed and the hybridprotein was purified to homogeneity in high yield. The chimericprotein was shown to have almost identical enzymatic characteristicsto pure 6-phospho-ß-galactosidase. This result leadsto the conclusion that a free N-terminus of the 6-phospho-ß-galactosidaseis not required for biological activity. The hybrid proteinof protein A and 6-phospho-ß-galactosidase was usedas an enzyme conjugate in enzyme-linked immunosorbent assays(ELISA). The experiments presented demonstrate that the 6-phospho-ß-galactosidaseis a suitable fusion partner in various diagnostic applicationswhere an unique biological activity is required.     

Native-like {beta}-hairpin structure in an isolated fragment from ferredoxin: NMR and CD studies of solvent effects on the N-terminal 20 residues

The conformational properties of protein fragments have beenwidely studied as models of the earliest initiation events inprotein folding. While native-like -helices and ß-turnshave been identified, less is known about the factors that underlyß-sheet formation, in particular ß-hairpins,where considerably greater long-range order is required. TheN-terminal 20 residue sequence of native ferredoxin I (fromthe blue-green alga Aphanothece sacrum ) forms a ß-hairpinin the native structure and has been studied in isolation byNMR and CD spectroscopy. Local native-like interactions aloneare unable to stabilize significantly a folded conformationof the 20-residue fragment in purely aqueous solution. However,we show that the addition of low levels of organic co-solventspromotes formation of native-like ß-hairpin structure.The results suggest an intrinsic propensity of the peptide toform a native-like ß-hairpin structure, and that theorganic co-solvent acts in lieu of the stabilizing influenceof tertiary interactions (probably hydrophobic contacts) whichoccur in the folding of the complete ferredoxin sequence. Thestructure of the isolated hairpin, including the native-likeregister of interstrand hydrogen bonding interactions, appearsto be determined entirely by the amino acid sequence. The solventconditions employed have enabled this intrinsic property tobe established.     

Modular mutagenesis of a TEM-barrel enzyme: the crystal structure of a chimeric E.coli TIM having the eighth {beta}{alpha}-unit replaced by the equivalent unit of chicken TIM

Abstract The crystal structure of a hybrid Escherichia coli triosephosphateisomerase (TIM) has been determined at 2.8 Å resolution.The hybrid TIM (ETIM8CHI) was constructed by replacing the eighthß-unit of E.coli TIM with the equivalent unit of chickenTIM. This replacement involves 10 sequence changes. One of thechanges concerns the mutation of a buried alanine (Ala232 instrand 8) into a phenylalanine. The ETIM8CHI structure showsthat the A232F sequence change can be incorporated by a side-chainrotation of Phe224 (in helix 7). No cavities or strained dihedralsare observed in ETIM8CHI in the region near position 232, whichis in agreement with the observation that ETIM8CHI and E.coliTIM have similar stabilities. The largest CA (C-alpha atom)movements, 3 Å, are seen for the C-terminal end of helix8 (associated with the outward rotation of Phe224) and for theresidues in the loop after helix 1 (associated with sequencechanges in helix 8). From the structure it is not clear whythe k_cat of ETIM8CHI is 10 times lower than in wild type E.coliTIM