Peter Brodin

Stimulation of intestinal basolateral membrane calcium-pump activity by recombinant synthetic calbindin-D9k and specific mutants

Calcium transport by the Ca2(+)-pumping ATPase in rat duodenal basolateral-enriched membrane vesicles was stimulated by synthetic calbindin-D9k in a similar fashion to the purified natural protein. In order to elucidate the mechanism of this effect, various synthetic mutant proteins were studied. Proteins with modifications to the N-terminal Ca2(+)-binding domain, or to a cluster of negatively-charged surface residues had altered Ca2(+)-binding but these changes did not affect the stimulation of vesicular Ca2+ transport. It appears that these domains are not essential for the interaction between calbindin-D9k and the intestinal basolateral Ca2(+)-pump.

High-yield purification of HIV-1 proteinase expressed by a synthetic gene in Escherichia coli

A rapid and simple purification procedure for human immunodeficiency virus type 1 (HIV-1) proteinase from a synthetic gene expressed in Escherichia coli has been developed. The synthetic gene was constructed from oligonucleotides containing several restriction enzyme sites in order to allow simple construction of homologous genes. The protein was translated as a precursor which was autocatalytically processed into the mature protein as shown by N-terminal sequence analysis of the purified protein. Immunoblot analysis was used to verify the nature of the expression product and it was found that 2 of 10 anti-peptide antibodies, covering the whole proteinase sequence, were able to react with the enzyme in crude bacterial lysates. These two anti-peptide antibodies represent a continuous sequence partially overlapping the active site. The purification involves two initial precipitation steps followed by cation-exchange and size-exclusion chromatography. A high yield and a high specific activity were achieved.

Neutralization of Surface Charges Markedly Affects the Properties of Bovine Calbindin D9k

Calbindin D9k constitutes an attractive model system for exploring the relationships between structure, dynamics and function in the EF-hand family of proteins. It is the smallest protein known (Mr ≈ 8,500; 75 a.a:s) with a pair of EF-hand Ca2+-binding sites and its structure resembles closely the globular domains of the homologous proteins calmodulin, parvalbumin and skeletal muscle troponin C. [1,7]. The schematic structure of calbindin is shown in Fig. 1.