Production and characterization of biologically active Ala-Ser-(His)6-Ile-Glu-Gly-Arg-human prolactin (tag-hPRL) secreted in the periplasmic space of Escherichia coli.

Human prolactin (hPRL) cDNA was obtained by screening of a pituitary cDNA library with a synthetic 21-mer oligonucleotide and with rat PRL cDNA. For its expression, use was made of a vector, p3SN8, containing tac-promoter-controlled sequences for a bacterial cellulase leader joined to sequences coding for Ala-Ser, a chromatographic affinity site consisting of six histidines and a Factor Xa cleavage site. The hPRL cDNA was inserted at the 3' end of the cleavage-site sequences. Expression in Escherichia coli led to secretion in the periplasmic space of a fully bioactive hPRL variant constituting authentic hPRL with a peptide tag, i.e. Ala-Ser-(His)6-Ile-Glu-Gly-Arg, at its N-terminal. This tag-hPRL could be rapidly and efficiently purified by metal-chelate affinity chromatography. The correct processing and quality of tag-hPRL was monitored by SDS/PAGE, Western-blot analysis, immunoassay and Nb2-lymphoma-cell bioassay. Treatment with Factor Xa for tag removal was only partially successful. Periplasmic secretion of tag-hPRL of the order of 0.7 micrograms/ml per A600 unit and one-step purification indicate feasibility for tag-hPRL production for in vitro diagnostic and research applications. This is the first report describing periplasmic secretion of a bioactive form of hPRL.

Analysis of disulphide bridge function in recombinant bovine prolactin using site-specific mutagenesis and renaturation under mild alkaline conditions: a crucial role for the central disulphide bridge in the mitogenic activity of the hormone.

We have previously described a method for isolating Escherichia coli-produced methionyl bovine prolactin (Met-bPRL) and its renaturation using thioredoxin. This report describes an alternative renaturation procedure in which extracted Met-bPRL is incubated in air at pH 10 and 20 degrees C. Within 1 h of such treatment essentially all of the reduced Met-bPRL was converted to the oxidized form; this was accompanied by an increase to full mitogenic activity in the Nb2 cell bioassay. It was also found that, to minimize contamination by high mol. wt Met-bPRL derivatives, it is essential to have a reducing agent (dithiothreitol) present during disruption of the bacteria and to extract the protein at neutral pH. The contribution of each of the three disulphide bridges in bPRL to its bioactivity was studied with Met-bPRL variants, prepared via site-specific mutagenesis, in which cysteines were replaced by serines to prevent disulphide bond formation. Variants lacking the C4-C11 bridge, the C191-C199 bridge or both these terminal bridges were as mitogenic as authentic bPRL. (Variants lacking the C191-C199 bridge had markedly increased solubility in the presence of deoxycholate.) In contrast, variants lacking the C58-C174 bridge had greatly reduced bioactivity, indicating that integrity of the large disulphide loop is crucial to the hormone's mitogenic activity.

Single amino acid substitutions in recombinant bovine prolactin that markedly reduce its mitogenic activity in Nb2 cell cultures.

Three amino acid residues of bovine PRL (bPRL) have been examined for their roles in the mitogenic activity of the hormone in Nb2 lymphoma cell cultures. The residues of interest, R21, R177, and K187, are conserved in eight pituitary PRLs, but not in the related, nonlactogenic bGH. Using site-specific mutagenesis, a number of recombinant methionyl bPRL variants have been prepared, each of which contained a single amino acid substitution of one of the three residues; a variety of amino acids was used for substitution. Twelve exchanges of R177 (to A, L, N, K, D, E, Y, G, S, Q, H, and F) all led to marked decreases in mitogenic activity. Even the conservative change, R177K, led to a decrease in mitogenic activity of about 90%; all the other R177 substitutions led to even more marked decreases; there was essentially complete loss of activity when the positively charged R177 was replaced by the negatively charged aspartate. Exchanges of R21 (to A, L, N, and K) were less dramatic, with the greatest decrease (79%) occurring in the case of R21A. Exchanges of K187 (to A, L, N, and R) had a relatively minor effect on the mitogenic activity of the hormone. Residues R21 and R177 in bPRL are located in putative helices 1 and 4, respectively; in the three-dimensional structure of the hormone these residues are predicted to be quite closely apposed. The results suggest that R177 and, to a lesser degree, R21 have important roles in the mitogenic activity of bPRL.

Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2+.

Ferric reductase activity was examined in Azotobacter vinelandii and was found to be located in the cytoplasm. The specific activities of soluble cell extracts were not affected by the iron concentration of the growth medium; however, activity was inhibited by the presence of Zn2+ during cell growth and also by the addition of Zn2+ to the enzyme assays. Intracellular Fe2+ levels were lower and siderophore production was increased in Zn2+-grown cells. The ferric reductase was active under aerobic conditions, had an optimal pH of approximately 7.5, and required flavin mononucleotide and Mg2+ for maximum activity. The enzyme utilized NADH to reduce iron supplied as a variety of iron chelates, including the ferrisiderophores of A. vinelandii. The enzyme was purified by conventional protein purification techniques, and the final preparation consisted of two major proteins with molecular weights of 44,600 and 69,000. The apparent Km values of the ferric reductase for Fe3+ (supplied as ferric citrate) and NADH were 10 and 15.8 microM, respectively, and the data for the enzyme reaction were consistent with Ping Pong Bi Bi kinetics. The approximate Ki values resulting from inhibition of the enzyme by Zn2+, which was a hyperbolic (partial) mixed-type inhibitor, were 25 microM with respect to iron and 1.7 microM with respect to NADH. These results suggested that ferric reductase activity may have a regulatory role in the processes of iron assimilation in A. vinelandii.

Characterization of the porins of Campylobacter jejuni and Campylobacter coli and implications for antibiotic susceptibility.

The major outer membrane protein was extracted from Campylobacter coli by Triton X-100/EDTA fractionation of cell envelopes. This heat-modifiable protein was shown to have pore-forming activity in black lipid bilayers. The C. coli porin formed a relatively small cation-selective pore with a mean single-channel conductance of 0.53 +/- 0.16 nS in 1.0 M KCl. There was no evidence of oligomer formation, which suggested that each protein monomer formed a pore. Pore-forming activity of the C. coli porin and similarly prepared Campylobacter jejuni porin was also measured in liposome-swelling assays. These results confirmed the cation selectivity of both pores. The C. coli porin formed a small pore, which hindered the penetration of solutes with a molecular weight of 262, and a larger pore, which hindered the penetration of solutes with a molecular weight of 340, in a protein-concentration-dependent manner. C. jejuni formed one size of pore that was slightly larger than the C. coli pore and just permitted the passage of solutes, with a molecular weight of 340. A review of the literature concerning in vitro screening of antimicrobial agents tended to confirm the low permeability of the C. jejuni outer membrane to hydrophilic antimicrobial agents except when the molecules had molecular weights of less than 360. The porins of C. jejuni and C. coli may contribute to intrinsic resistance to antimicrobial agents, whereas alternative (nonporin) routes of antimicrobial agent uptake may be more important determinants of susceptibility to antimicrobial agents.

Zn Increases Siderophore Production in Azotobacter vinelandii.

When Azotobacter vinelandii was grown in the presence of low levels of iron, the addition of 20 or 40 muM ZnSO(4) caused earlier production of the catechol siderophores and a dramatic increase in the amount of azotobactin. The level of cellular iron was not significantly lowered in Zn -grown cells, which suggested that Zn was not causing more severe, or earlier, iron limitation. Also, Zn did not appear to affect production of the high-molecular-weight outer membrane iron-repressible proteins that presumably function as ferrisiderophore receptors. Spectrophotometric examination of ion binding to the siderophores revealed that while the siderophores appeared to bind Zn, only in the case of azotochelin was iron unable to completely overcome any Zn -induced changes in the absorption spectra. This appeared to rule out direct competition of Zn with iron for binding to the siderophores. Fe uptake was depressed both in Zn -grown cells and in Zn -free cells to which Zn was added during the uptake assay, except with azotobactin, with which the level of Fe uptake by Zn -grown cells was close to control levels. These results suggested two possible sites where Zn could be acting, one involving the biosynthesis of siderophores and possibly the genetic regulation of the iron assimilation system and the other involving an internal point common to iron assimilation by both high- and low-affinity iron uptake.

Derepression of the Azotobacter vinelandii siderophore system, using iron-containing minerals to limit iron repletion.

Azotobacter vinelandii solubilized iron from certain minerals using only dihydroxybenzoic acid, which appeared to be produced constitutively. Solubilization of iron from other minerals required dihydroxybenzoic acid and the siderophore N,N'-bis-(2,3- dihydroxybenzoyl )-L-lysine ( azotochelin ) or these chelators plus the yellow-green fluorescent siderophore azotobactin . In addition to this sequential production of siderophores, cells also demonstrated partial to hyperproduction relative to the iron-limited control. The iron sources which caused partial derepression of the siderophores caused derepression of all the high-molecular-weight iron-repressible outer membrane proteins except a 77,000-molecular-weight protein, which appeared to be coordinated with azotobactin production. Increased siderophore production correlated with increased production of outer membrane proteins with molecular weights of 93,000, 85,000, and 77,000, but an 81,000-molecular-weight iron-repressible protein appeared at a constant level despite the degree of derepression. When iron was readily available, it appeared to complex with a 60,000-molecular-weight protein believed to form a surface layer on the A. vinelandii cell.