| 1. |
- Nilsson, J, et al.
(författare)
-
Competitive elution of protein A fusion proteins allows specific recovery under mild conditions.
- 1994
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 224:1, s. 103-8
-
Tidskriftsartikel (refereegranskat)abstract
- A novel system is described for mild elution of fusion proteins by competitive elution. The approach is based on displacement of immobilized fusions containing a monovalent IgG-binding staphylococcal protein A fragment (Z) from an IgG-affinity matrix by a divalent fragment fused to a serum-albumin-binding region derived from streptococcal protein G. Using real-time interaction analysis, the binding (K(aff)) to polyclonal human IgG was found to be 3.3 (+/- 0.4) x 10(8) M-1 for divalent ZZ and 2.0 (+/- 0.1) x 10(7) M-1 for monovalent Z. This more than tenfold difference in binding strength ensures a high efficiency in the elution step. The competitor protein can specifically be removed and recovered from the elution mixture by subsequent passage through a human serum albumin(HSA)-affinity column, leaving only the target fusion protein in the flow-through fraction. Here, we show that a recombinant Klenow fragment of DNA polymerase I expressed in Escherichia coli can be recovered with high yield, and retained activity, from a crude bacterial lysate by IgG-affinity chromatography using mild conditions during both binding and elution.
|
|
| 2. |
- Bitzén, Ulrika, et al.
(författare)
-
Retinyl palmitate is a reproducible marker for chylomicron elimination from blood
- 1994
-
Ingår i: Scandinavian Journal of Clinical & Laboratory Investigation. - : Informa UK Limited. - 1502-7686 .- 0036-5513. ; 54:8, s. 611-613
-
Tidskriftsartikel (refereegranskat)abstract
- To study the individual variation in chylomicron clearance rate, young healthy volunteers were given a p.o. dose of 50,000 IU retinyl palmitate in the morning to label their chylomicrons. Serial blood samples were then obtained in the time interval 4-8 h after retinyl palmitate intake, to closely monitor the clearance of retinyl ester from the blood. The procedure was repeated in an identical way two days later. The calculated individual halflives for retinyl palmitate clearance ranged from 1.54 to 9.90 h, i.e. a more than five-fold variation. The intraindividual variation was much less (relative SD 11%). Retinyl palmitate clearance (and probably chylomicron clearance) is, thus, relatively constant within the same individual on different occasions but varies considerably between individuals.
|
|
| 3. |
- Spyrou, Giannis, et al.
(författare)
-
Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme
- 1991
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 173:12, s. 3673-3679
-
Tidskriftsartikel (refereegranskat)abstract
- The enzyme NAD(P)H:flavin oxidoreductase (flavin reductase) catalyzes the reduction of soluble flavins by reduced pyridine nucleotides. In Escherichia coli it is part of a multienzyme system that reduces the Fe(III) center of ribonucleotide reductase to Fe(II) and thereby sets the stage for the generation by dioxygen of a free tyrosyl radical required for enzyme activity. Similar enzymes are known in other organisms and may more generally be involved in iron metabolism. We have now isolated the gene for the E. coli flavin reductase from a lambda gt11 library. After DNA sequencing we found an open reading frame coding for a polypeptide of 233 amino acids, with a molecular weight of 26,212 and with an N-terminal segment identical to that determined by direct Edman degradation. The coding sequence is preceded by a weak ribosome binding site centered 8 nucleotides from the start codon and by a promoterlike sequence centered at a distance of 83 nucleotides. In a Kohara library the gene hybridized to position 3680 on the physical map of E. coli. A bacterial strain that overproduced the enzyme approximately 100-fold was constructed. The translated amino acid sequence contained a potential pyridine nucleotide-binding site and showed 25% identity with the C-terminal part of one subunit (protein C) of methane monooxygenase from methanotropic bacteria that reduces the iron center of a second subunit (protein A) of the oxygenase by pyridine nucleotides.
|
|
