Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli

Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E. coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism. The plant ACP expressed in E. coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP. We have purified and characterized the recombinant spinach ACP-I. NH2-terminal amino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine. Recombinant ACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I. Recombinant ACP-I was a poor substrate for E. coli fatty acid synthesis. In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E. coli ACP. Similarly, malonyl-coenzyme A:acyl carrier protein transacylase isolated from E. coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B. napus reacted equally well with either E. coli ACP or recombinant ACP-I. E. coli acyl-ACP synthetase showed a higher reaction rate for recombinant ACP-I than for E. coli ACP. Expression of spinach ACP-I in E. coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependent reactions of plant lipid metabolism.     

Expression of an active spinach acyl carrier protein-I/protein-A gene fusion

A synthetic gene encoding spinach acyl carrier protein I (ACP-I) was fused to a gene encoding the Fc-binding portion of staphylococcal protein A. This gene fusion, under the control of the P_R promoter, was expressed at high levels in Escherichia coli producing a 42 kDa fusion protein. This fusion protein was phosphopantethenylated in E. coli. In vitro the ACP portion of the fusion protein was able to participate in acyl ACP synthetase reactions, plant malonyl-CoA:ACP transacylase (MCT) reactions, and plant fatty acid synthetase (FAS) reactions. Inhibitory effects of high ACP concentrations on in vitro plant FAS were observed with the unfused ACP-1 but not with the fusion protein. As with unfused ACP-I, the fusion protein was a poor substrate for E. coli FAS reactions. When injected into rabbits, the fusion protein was also able to generate antiserum to spinach ACP-I.     

The recombinant spinach acyl-acyl carrier protein-I expressed in Escherichia coli is the 18:1 delta 11(cis) thioester

A synthetic spinach acyl carrier protein-I (ACP-I) gene was cloned and expressed in the Escherichia coli beta-alanine auxotroph SJ16 (P. D. Beremand et al. (1987) Arch. Biochem. Biophys. 256, 90-100). After characterization of the transformed cells and purification of the protein product it was evident that 50% of the recombinant spinach ACP-I was acylated during early log-phase growth (D. J. Guerra et al. (1988) J. Biol. Chem. 263, 4386-4391). We have purified the recombinant acyl-acyl carrier protein-I to greater than 90% homogeneity and have made a fatty acid methyl ester of the delipidated and trypsin-treated preparation. We have found that the acyl moiety attached to recombinant spinach acyl carrier protein-I is 18:1 delta 11(cis) (cis-vaccenic acid) a major unsaturated end product of Escherichia coli de novo fatty acid synthesis. This result reflects previous work (D. S. Guerra et al. (1986) Plant Physiol. 82, 448-453) which suggested the acyl carrier protein-I structure has evolved from ancestral ACP structures to accommodate the eukaryotic pathway of lipid synthesis in higher plants. The accumulation of recombinant 18:1 delta 11(cis) acyl carrier protein-I in transformed E. coli SJ16 cells attests to the poor reactivity of this substrate to acyl transferase reactions and may help explain the lack of effect on pools of fatty acids found in vivo.     

Site-directed mutagenesis of the spinach acyl carrier protein-I prosthetic group attachment site

Site-directed mutagenesis was used to change the phosphopantetheine attachment site (Ser38) of spinach acyl carrier protein I (ACP-I) from a serine to a threonine or cysteine residue. 1. Although the native ACP-I is fully phosphopantethenylated when expressed in Escherichia coli, the TH-ACP-I and CY-ACP-I mutants were found to be completely devoid of the phosphopantetheine group. Therefore, the E. coli holoACP synthase requires serine for in vivo phosphopantetheine addition to spinach ACP-I. 2. Spinach holoACP synthase was completely inactive in vitro with either the TH-ACP-I or CY-ACP-I mutants. In addition, TH-ACP-I and CY-ACP-I were strong inhibitors of spinach holoACP synthase. 3. The mutant ACPs were weak or ineffective as inhibitors of spinach fatty acid synthesis and spinach oleoyl-ACP hydrolase. 4. Compared to holoACP-I, the mutant apoACP-I analogs had: (a) altered mobility in SDS and native gel electrophoresis, (b) altered binding to anti-(spinach ACP-I) antibodies and (c) altered isoelectric points. The combined physical, immunological and enzyme inhibition data indicate that attachment of the phosphopantheine prosthetic group alters ACP conformation.     

Expression of holo and apo forms of spinach acyl carrier protein-I in leaves of transgenic tobacco plants.

Acyl carrier protein (ACP) is a chloroplast-localized cofactor of fatty acid synthesis, desaturation, and acyl transfer. We have transformed tobacco with a chimeric gene consisting of the tobacco ribulose-1,5-bisphosphate carboxylase promoter and transit peptide and the sequence encoding the mature spinach ACP-I. Spinach ACP-I was expressed in the transformed plants at levels twofold to threefold higher than the endogenous tobacco ACPs as determined by protein immunoblots and assays of ACP in leaf extracts. In addition to these elevated levels of the holo form, there were high levels of apoACP-I, a form lacking the 4'-phosphopantetheine prosthetic group and not previously detected in vivo. The mature forms of both apoACP-I and holoACP-I were located in the chloroplasts, indicating that the transit peptide was cleaved and that attachment of the prosthetic group was not required for uptake into the plastid. There were also significant levels of spinach acyl-ACP-I, demonstrating that spinach ACP-I participated in tobacco fatty acid metabolism. Lipid analyses of the transformed plants indicated that the increased ACP levels caused no significant alterations in leaf lipid biosynthesis.     

Hepatic microsomal short-chain beta-hydroxyacyl-CoA dehydrase distinct from the fatty acid elongation component: substrate specificity of the membrane-extracted enzyme

The ability of 0.4 M KCl to extract over 80% of a short-chain beta-hydroxyacyl-CoA dehydrase from rat hepatic endoplasmic reticulum, while more than 80% of the long-chain beta-hydroxyacyl-CoA dehydrase component of the fatty acid chain elongation system remains intact, confirms the existence of more than one hepatic microsomal dehydrase. Following extraction from the microsomal membrane, the short-chain dehydrase undergoes, at least, a two-fold activation. Employing even-numbered trans-2-enoyl-CoA substrates ranging in carbon chain length from 4 to 16, the highest dehydrase specific activity of 16 mumol min-1 mg protein-1 was obtained with trans-2-hexenoyl-CoA; crotonyl-CoA was the second most active substrate, followed by 8 greater than 10 greater than 12 greater than 14 greater than 16. The specific activity of the short-chain dehydrase with trans-2-hexadecenoyl-CoA (C-16) was only 3% of that observed with the trans-2-hexenoyl-CoA. With crotonyl-CoA or beta-hydroxybutyryl-CoA as substrates, HPLC was employed to identify the products, beta-hydroxybutyryl-CoA, of the hydration reaction, or crotonyl-CoA, of the reverse dehydration reaction. It was also observed that the short-chain dehydrase catalyzed the formation of both D(-) and L(+) stereoisomers of beta-hydroxybutyryl-CoA. The equilibrium constant for the dehydrase-catalyzed reaction determined at pH 7.4 and 35 degrees C, was calculated to be 6.38 X 10(-2) M-1, while the standard free energy change was -775 cal/mol, results similar to those obtained with crystalline crotonase. Finally, based on membrane fraction marker enzymes, substrate specificity, and heat lability of the dehydrase, it was concluded that the microsomal membrane contains a short-chain beta-hydroxyacyl-CoA dehydrase which is separate from the mitochondrial crotonase.     

Isolation of the Rhizobium leguminosarum NodF nodulation protein: NodF carries a 4''-phosphopantetheine prosthetic group.

Rhizobium species produce a protein product of the nodF gene that has a limited but recognizable homology to the well-characterized acyl carrier protein (ACP) of Escherichia coli. NodF functions together with NodE in generating a host-specific response to the plant host in the interchange of signals leading to the effective nodulation of roots (H.P. Spaink, J. Weinman, M.A. Djordjevic, C.A. Wijffelman, R.J.H. Okker, and B. J.J. Lugtenberg, EMBO J. 8:2811-2818, 1989; B. Scheres, C. van de Wiel, A. Zalensky, B. Horvath, H. Spaink, H. van Eck, F. Zwartkruis, A.M. Wolters, T. Gloudemans, A. van Kammen, and T. Bisseling, Cell 60:281-294, 1990). The nodFE region of Rhizobium leguminosarum has been cloned into a multicopy plasmid and has been shown in R. leguminosarum to code for a flavonoid-inducible protein that is effectively labeled by radioactive beta-alanine added to the growth medium. After purification, the labeled protein migrates as a single band with an apparent molecular weight of 5,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, more rapidly than E. coli ACP. In contrast, in native gels the protein is resolved into two bands, both identified as NodF by analysis of the amino terminus and both migrating more slowly than E. coli ACP. Pulse-chase experiments with labeled beta-alanine suggested that the slower-moving band may be the precursor of the faster band. The NodF protein carries a 4'-phosphopantetheine as a prosthetic group. A NodF fusion protein under the control of the lac promoter is expressed in E. coli and is labeled with beta-alanine, indicating that it is recognized by the ACP synthase of E. coli. The ACP phosphodiesterase of E. coli, which catalyzes the release of phosphopantetheine from E. coli ACP, does not remove phosphopantetheine from NodF.     

The enrichment of a ruminal bacterium (Megasphaera elsdenii YJ-4) that produces the trans-10, cis-12 isomer of conjugated linoleic acid

AIMS: To isolate predominant ruminal bacteria that produce trans-10, cis-12 conjugated linoleic acid (CLA) from linoleic acid (LA). METHODS AND RESULTS: Mixed bacteria from ruminal contents of a cow fed grain were enriched with DL-lactate and trypticase. They produced more trans-10, cis-12 CLA than those that were not enriched (7 vs 2 microg mg protein(-1), P < 0.05). Enrichments had an abundance of large cocci that produced trans-10, cis-12 CLA from LA. Strain YJ-4 produced the most trans-10, cis-12 CLA (approx. 7 microg mg protein(-1)) and 16S rDNA sequencing indicated that YJ-4 was a strain of Megasphaera elsdenii. Megasphaera elsdenii T81 produced approx. 4 microg trans-10, cis-12 CLA mg protein(-1) while strains B159, AW106 and JL1 produced < 0.5 microg mg protein(-1). The trans-10, cis-12 CLA production of YJ-4 was first order with respect to cell concentration (0-800 microg protein ml(-1)), but kinetics were not first order with respect to substrate concentration. CONCLUSIONS: Some M. elsdenii strains produce significant amounts of trans-10, cis-12 CLA. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-10, cis-12 CLA appears to cause milk fat depression in cattle fed diets supplemented with grain and polyunsaturated fatty acids, but predominant ruminal bacteria that produced trans-10, cis-12 CLA from LA had not previously been isolated.     

A new mechanism for anaerobic unsaturated fatty acid formation in Streptococcus pneumoniae

The anaerobic pathway for unsaturated fatty acid synthesis was established in the 1960s in Escherichia coli. The double bond is introduced into the growing acyl chain by FabA, an enzyme capable of both the dehydration of beta-hydroxydecanoyl-acyl carrier protein (ACP) to trans-2-decenoyl-ACP, and the isomerization of trans-2 to cis-3-decenoyl-ACP. However, there are a number of anaerobic bacteria whose genomes do not contain a fabA homolog, although these organisms nonetheless produce unsaturated fatty acids. We cloned and biochemically characterized a new enzyme in type II fatty acid synthesis from Streptococcus pneumoniae that carries out the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP, but is not capable of catalyzing the dehydration of beta-hydroxy intermediates. This tetrameric enzyme, designated FabM, has no similarity to FabA, but rather is a member of the hydratase/isomerase superfamily. Thus, the branch point in the biosynthesis of unsaturated fatty acids in S. pneumoniae occurs following the formation of trans-2-decenoyl-ACP, in contrast to E. coli where the branch point takes place after the formation of beta-hydroxydecanoyl-ACP.     

Photocontrol of gibberellin metabolism in situ in maize

Two forms of spinach acyl carrier protein (ACP-I and ACP-II) have recently been characterized and found to be expressed in a tissue-specific manner (JB Ohlrogge, TM Kuo, 1985 J Biol Chem 260: 8032). To examine possible different functions for these ACP isoforms, we have tested purified preparations of spinach leaf ACP-I and ACP-II and Escherichia coli ACP in several in vitro reactions of fatty acid metabolism. Total de novo fatty acid synthesis and malonyl-CoA:ACP transacylase do not appear to discriminate between acyl carrier protein isoforms. In contrast, the K_m of oleoyl-ACP thioesterase for oleoyl-ACP-II is 10-fold higher than for oleoyl-ACP-I, whereas the K_m of acyl-ACP glycerol-3-phosphate acyl transferase is 5-fold higher for oleoyl-ACP-I than for oleoyl-ACP-II. A characterization of these reactions and a possible role for ACP isoforms in regulation of fatty acid metabolism in plants are described.     

Spinach holo-acyl carrier protein: overproduction and phosphopantetheinylation in Escherichia coli BL21(DE3), in vitro acylation, and enzymatic desaturation of histidine-tagged isoform I

Spinach ACP isoform I was overexpressed in Escherichia coli BL21(DE3) using a gene synthesized from codons associated with high-level expression in E. coli. The synthetic gene has extensive changes in codon usage (23 of 77 total codons) relative to that of the originally synthesized plant gene (P. D. Beremand et al., 1987, Arch. Biochem. Biophys. 256, 90-100). After expression of the new synthetic gene, purified ACP and ACP-His6 were obtained in yields of up to 70 mg L-1 of culture medium, compared to approximately 1-6 mg L-1 of purified ACP obtained from the gene composed of predicted spinach codons. In either shaken flask or fermentation culture, approximately 15% conversion to holo-ACP or holo-ACP-His6 was obtained regardless of the level of protein expression. However, coexpression of ACP-His6 with E. coli holo-ACP synthase in E. coli BL21(DE3) during pH- and dissolved O2-controlled fermentation routinely yielded greater than 95% conversion to holo-ACP-His6. Electrospray ionization mass spectrometric analysis of the purified recombinant ACPs revealed that the amino terminal Met was efficiently removed, but only if the bacterial cell lysates were prepared in the absence of EDTA. This observation is consistent with the inhibition of endogenous Met-aminopeptidase by removal of catalytically essential Co(II) and introduces the importance of considering the catalytic properties of host enzymes providing ad hoc posttranslational modification of recombinant proteins. Stearoyl-ACP-His6 was shown to be indistinguishable from stearoyl-ACP as a substrate for enzymatic acylation and desaturation. In combination, these studies provide a coordinated scheme to produce and characterize quantities of acyl-ACPs sufficient to support expanded biophysical and structural studies.     

The role of Delta(9)-desaturase in the production of cis-9, trans-11 CLA

Biomedical studies with animal models have demonstrated that cis-9, trans-11 conjugated linoleic acid (CLA), the predominant isomer found in milk fat from dairy cows, has anticarcinogenic effects. We recently demonstrated endogenous synthesis of cis-9, trans-11 CLA from ruminally derived trans-11 C18:1 by Delta(9)-desaturase in lactating dairy cows. The present study further examined endogenous synthesis of cis-9, trans-11 CLA and quantified its importance by increasing substrate supply using partially hydrogenated vegetable oil (PHVO) as a source of trans-11 C18:1 and blocking endogenous synthesis using sterculic oil (SO) as a source of cyclopropene fatty acids which specifically inhibit Delta(9)-desaturase. Four cows were abomasally infused with 1) control, 2) PHVO, 3) SO, and 4) PHVO+SO in a 4 x 4 Latin square design. With infusion of PHVO, cis-9, trans-11 CLA was increased by 17% in milk fat. Consistent with inhibition of desaturase, SO treatments increased milk fat ratios for the fatty acid pairs effected by Delta(9)-desaturase, C14:0/cis-9 C14:1, C16:0/cis-9 C16:1, and C18:0/cis-9 C18:1. The role of endogenous synthesis of CLA was evident from the 60-65% reduction in cis-9, trans-11 CLA which occurred in milk fat with SO treatments. cis-9 C14:1 originates from desaturation of C14:0 by Delta(9)-desaturase and can be used to estimate the extent of SO inhibition of Delta(9)-desaturase. When this correction factor was applied, endogenous synthesis was estimated to account for 78% of the total cis-9, trans-11 CLA in milk fat. Thus, endogenous synthesis was the major source of cis-9, trans-11 CLA in milk fat of lactating cows.     

Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.     

A double role for a strictly conserved serine: further insights into the dUTPase catalytic mechanism

Ser72 at the active site of the Escherichia coli dUTPase has been mutated to an alanine, and the properties of the mutant have been investigated. The serine is absolutely conserved among the monomeric and trimeric dUTPases (including the bifunctional dCTP deaminase:dUTPases), and it has been proposed to promote catalysis by balancing negative charge at the oxygen that bridges the alpha- and beta-phosphorus of the substrate. In all reported complexes of dUTPases with the substrate analogue alpha,beta-imido-dUTP.Mg, the serine beta-OH is indeed hydrogen bonded to the alpha,beta-bridging nitrogen of the analogue. However, in the complex of the Asp90 --> Asn mutant dUTPase with the true substrate dUTP.Mg, the serine beta-OH points in the opposite direction and may form a hydrogen bond to Asn84 at the bottom of the pyrimidine pocket. Here we show that the replacement of the beta-OH by hydrogen reduces k cat from 5.8 to 0.008 s (-1) but also k -1 , the rate of substrate dissociation, from 6.2 to 0.1 s (-1) ( K M = 6 x 10 (-9) M). We conclude that the serine beta-OH exercises both ground state (GS) destabilization and transition state (TS) stabilization, effects not usually linked to a single residue. With experimental support, we argue that the beta-OH destabilizes the GS by imposing conformational constraints on the enzyme and that formation of the TS depends on a rotation of the serine side chain that not only relieves the constraints but brings the beta-OH into a position where it can electrostatically stabilize the TS. This rotation would also allow the beta-OH to promote both deamination and hydrolysis in the bifunctional deaminases. We find that the E. coli dUTPase does not catalyze the hydrolysis of the alpha,beta-imido-dUTP.Mg, suggesting that the analogue provides the hydrogen in the bond to the serine beta-OH.     

Identification of a key residue in the conformational stability of acyl carrier protein

Conformational flexibility of acyl carrier protein (ACP) is important for its ability to interact with multiple enzymes in bacterial fatty acid metabolism. We have recently shown that, unlike the prototypical ACP from Escherichia coli, the more acidic Vibrio harveyi ACP is largely unfolded at physiological pH. Mutations D18K, A75H and A75H/D18K were made in recombinant V. harveyi ACP (rACP) to determine the importance of basic residues Lys-18 and His-75 in maintaining the native conformation of E. coli ACP. Both D18K and A75H ACPs were fatty acylated by acyl-ACP synthetase, showing that neither mutation grossly alters tertiary structure. Circular dichroism (CD) indicated that rACP refolded upon addition of MgCl(2) at 100-fold lower concentrations (<1 mM) than KCl, suggesting that divalent cations stabilize rACP by interaction at specific sites. Surprisingly, mutants A75H and A75H/D18K exhibited native-like conformation in the absence of MgCl(2), while the D18K mutant was comparable to rACP. Moreover, the alpha-helical content of A75H, A75H/D18K and E. coli ACPs was more sensitive than that of rACP or D18K ACP to modification by the histidine-selective reagent diethylpyrocarbonate. Together, these results suggest that the partial positive charge of His-75 may be important in maintaining the conformational stability of E. coli ACP at a neutral pH.     

Antiobesity effect of trans-10,cis-12-conjugated linoleic acid-producing Lactobacillus plantarum PL62 on diet-induced obese mice

AIMS: To observe the antiobesity activity of trans-10,cis-12-conjugated linoleic acid (CLA)-producing lactobacillus in mice. METHODS AND RESULTS: Lactobacillus plantarum PL62, which can grow in the presence of linoleic acid, was selected and studied. The culture supernatant of Lact. plantarum PL62 contained trans-10,cis-12-conjugated linoleic acid (6.4 microg ml(-1)), and the crude enzyme prepared from washed cells produced trans-10,cis-12 CLA (1395 microg mg(-1) protein). Lact. plantarum PL62 reduced the weights of epididymal, inguinal, mesenteric, and perirenal white adipose tissues and significantly reduced the blood levels of total glucose and body weights of mice (P<0.01). CONCLUSIONS: trans-10,cis-12-CLA-producing Lact. plantarum PL62 can exert the same antiobesity activity as trans-10,cis-12-CLA in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: trans-10,cis-12-CLA-producing Lactobacillus can be a replacement for CLA for obesity treatment via the continuous production of trans-10,cis-12-CLA. The results provide a novel opportunity to develop foods with antiobesity activity.     

Structure-activity relationship of conjugated linoleic acid and its cognates in inhibiting heparin-releasable lipoprotein lipase and glycerol release from fully differentiated 3T3-L1 adipocytes

Conjugated linoleic acid (CLA) reduces body fat in part by inhibiting the activity of heparin-releasable lipoprotein lipase (HR-LPL) activity in adipocytes, an effect that is induced by the trans-10,cis-12 CLA isomer. In this study we used a series of compounds that are structurally related to CLA (i.e., CLA cognates) to investigate the structural basis for this phenomenon. None of the 18:1 CLA cognates that were tested, nor trans-9,cis-12 18:2, cis-12-octadecen-10-ynoic acid (10y,cis-12) or 11-(2'-(n-pentyl)phenyl)-10-undecylenic acid (designated P-t10), exhibited any significant effect on HR-LPL activity. Among the CLA derivatives (alcohol, amide, and chloride) that were tested, only the alcohol form inhibited HR-LPL activity, although to a lesser extent than CLA itself. In addition, intracellular TG was reduced only by trans-10,cis-12 CLA and the alcohol form of CLA. Hence it appears that the trans-10,cis-12 conjugated double bond in conjunction with a carboxyl group at C-1 is required for inhibition of HR-LPL activity, and that an alcohol group can partially substitute for the carboxyl group. We also studied glycerol release from the cells, observing that this was enhanced by trans-10 18:1, trans-13 18:1, cis-12 18:1, cis-13 18:1, P-t10 but was reduced by cis-9 18:1, the alcohol and amide forms of CLA or 10y,cis-12. Accordingly the structural feature or features involved in regulating lipolysis appear to be more complex. Despite enhancing lipolysis in cultured 3T3-L1 adipocytes, trans-10 18:1 did not reduce body fat gain when fed to mice.     

The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli

Abstract: The production of the fusion protein staphylococcal protein A/E. coli β-galactosidase in Escherichia coli was studied in batch and fed batch cultivations. Batch cultivation of a recombinant E. coli strain yielded a final cell dry weight of 16.4 g 1^-1 with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the cell dry weight. Fed batch cultivation made it possible to increase the final cell dry weight to 77.0 g 1^-1. The intracellular product concentration (25%) was lower as compared to batch cultivation resulting in a total concentration of recombinant protein of 19.2 g 1^-1.     

The primary structure of spinach acyl carrier protein

Acyl carrier protein (ACP) from spinach leaves has been purified to homogeneity by high-performance liquid chromatography with an anion-exchange column. The amino acid sequence of one major ACP in spinach leaves, ACP-I, has been determined by automated Edman degradation. It consists of the following 82 amino acids: (sequence in text). Sequencing of the intact polypeptide provided data for the first 57 residues. Cleavage of the succinylated ACP with CNBr at Met-46, followed by sequencing of the fragment mixture, provided data for the final 36 residues. The C-terminal alanine was confirmed by carboxypeptidase Y digestion. The spinach ACP has 40, 70, and 25% homology with Escherichia coli, barley, and rabbit ACPs, respectively. The results not only provide the first complete sequence of a plant ACP, but also provide insight into the structural and evolutionary relationships among plant, animal, and bacterial ACPs.     

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.