N-terminal Aa Sequence Research Articles

Purification of a novel hydrophobin PN1 involved in antibacterial activity from an edible mushroom Pleurotus nebrodensis

Due to their hydrophobic characteristics, hydrophobins function in a broad range of processes in the growth and development of filamentous fungi. In this study, a novel hydrophobin, PN1, was purified from the fruiting bodies of Pleurotus nebrodensis by first removing other proteins from mycelial cell walls with hot SDS solution. The molecular mass of PN1 was approximately 10.7 kDa. The N-terminal AA sequence was QESPVNQCNTGSIEECKTVQ, which did not match that of any other mushroom hydrophobin described thus far. Levels of hydrophobin PN1 protein in the tissue of fruiting bodies was decreased by fruiting body development under low temperatures. Low levels of hydrophobin PN1 was correlated to the formation of water-soaked blotches on fruiting body tissues that appeared dark and translucent, and were usually sunken. In addition, hydrophobin PN1 levels were correlated with protecting fruiting bodies against surface bacterial colonization and multiplication. Low levels of hydrophobin PN1 resulted in water-soaked blotches that had high levels of surface bacteria belonging to multiple species when compared with nonsymptomatic tissue of fruiting bodies with higher hydrophobin PN1. These results show a direct link between mushroom hydrophobins and resistance of mushrooms to bacterial attack.

Erratum to: Purification and characterization of heterologously expressed nitrilases from filamentous fungi

Two works cited in the previous article (Kaplan et al. 2006, 2011) were corrected (Kaplan et al. 2013a) or retracted (Kaplan et al. 2013b). The reason was the falsity of data (N-terminal aa sequence and theMALDI-TOF analysis) by KBwhose ethics of scientific workwas examined in 2012 by the Ethics Commission of the Charles University in Prague and the Institute of Microbiology of the Academy of Sciences of the Czech Republic. The Commission found scientific misconduct of KB in several cases. Therefore, we wish to correct the following parts of the previous article: Introduction: “The nitrilase studied by us in Aspergillus niger K10 was the first fungal nitrilase to be purified from both its native... and heterologous producer...” Actually, the enzymes purified from the native and heterologous producer were different. The enzyme natively expressed in this strain was purified and characterised (Kaplan et al. 2006) and its partial aa sequence was recently published (Kaplan et al. 2013a). The recombinant enzyme was recently examined for its substrate specificity in whole cells (Kaplan et al. 2013c), and was found to be a cyanide hydratase with a significant nitrilase activity. Materials and methods, Nitrilase expression: Construction of plasmid pOK101 referred toKaplan et al. 2011 (retracted article). Therefore, we wish to describe this method here: To construct plasmid pOK101, NdeI-HindIII fragment of the PCR product amplified from cDNA of Aspergillus niger K10 (forward primer 5′-GCC ATATGG CAC CMG TCT TRA AGA AGTACA A3′, M=A or C; R=A or G, and reverse primer, 5′-GCA AGC TTT TAC TAG TTC TCC GAATCC ACG GT-3′) was ligated into vector pET-30a(+) (Novagen). Results, Variability of fungal nitrilases: The statement that in the nitrilase fromA . niger K10 “both its amino acid sequence and its biochemical properties have been already known” referred to Kaplan et al. 2011 (retracted article). For substrate specificity of this enzyme please refer to Kaplan et al. 2013c (see also above). Results, Nitrilase in Aspergillus niger K10: The hypothesis that the difference of substrate specificities in the natively expressed and the recombinant enzymes were caused by misfolding of the latter referred to Kaplan et al. 2011 (retracted article). Actually, the two enzymes were different in terms of their primary structure (see above). The online version of the original article can be found at http://dx.doi.org/ 10.1007/s00253-011-3525-7.

Purification and partial characterization of canine S100A12

Canine S100A12 (cS100A12) is a calcium-binding protein of the S100 superfamily of EF-hand proteins, and its expression is restricted to neutrophils and monocytes. Interaction of S100A12 with the receptor for advanced glycation end products (RAGE) has been suggested to play a central role in inflammation. Moreover, S100A12 has been shown to represent a sensitive and specific marker for gastrointestinal inflammation in humans. Only human, porcine, bovine, and rabbit S100A12 have been purified to date, and an immunoassay for the quantification of S100A12 is available only for humans. Therefore, the aim of this study was to develop a protocol for the purification of S100A12 and to partially characterize this protein in the dog ( Canis lupus familiaris) as a prelude to the development of an immunologic method for its detection and quantification in canine serum and fecal specimens. Leukocytes were isolated from canine whole blood by dextran sedimentation, and canine S100A12 was extracted from the cytosol fraction of these cells. Further purification of cS100A12 comprised of ammonium sulfate precipitation, hydrophobic interaction chromatography, and strong cation- and anion-exchange column chromatography. Canine S100A12 was successfully purified from canine whole blood. The relative molecular mass of the protein was estimated at 10,379.5 and isoelectric focusing revealed an isoelectric point of 6.0. The approximate specific absorbance of cS100A12 at 280 nm was determined to be 1.78 for a 1 mg/ml solution. The N-terminal AA sequence of the first 15 residues of cS100A12 was Thr-Lys-Leu-Glu-Asp-His-X-Glu-Gly-Ile-Val-Asp-Val-Phe-His, and revealed 100% identity with the predicted protein sequence available through the canine genome project. Sequence homology for the 14 N-terminal residues identified for cS100A12 with those of feline, bovine, porcine, and human S100A12 was 78.6%. We conclude that canine S100A12 can be successfully purified from canine whole blood using the described methods.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.

Prodefensins are matrix proteins of specific granules in human neutrophils

Defensins are potent antimicrobial and proinflammatory peptides. The human neutrophil defensins human neutrophil peptide (HNP)-1-3 are synthesized as 94 amino acide (aa) preproHNPs, which are converted to 75 aa proHNPs by cotranslational removal of a 19 aa endoplasmic reticulum signal peptide. At the promyelocytic stage of myelopoiesis, proHNPs are further proteolytically modified and accumulate in azurophil granules as 29-30 aa HNPs. In contrast, proHNPs produced by more mature myeloid cells are not subjected to proteolytic cleavage and undergo a high degree of constitutive exocytosis. The proHNPs are devoid of antimicrobial potential, and the significance of their secretion is unknown. To investigate whether mature neutrophils contain proHNPs, we developed antibodies against proHNP-1 by DNA immunization of rabbits. In addition, antibodies against the 45 aa proHNP pro-piece were raised by conventional immunization procedures. These antibodies allowed detection of proHNPs in homogenates of peripheral blood neutrophils. The proHNPs were isolated by affinity chromatography, and their identity was confirmed by mass spectrometry and N-terminal aa sequence analysis. Finally, the neutrophil proHNPs were identified as novel matrix proteins of specific granules by subcellular fractionation experiments, release studies, and immunoelectron microscopy. Thus, human neutrophils not only store large amounts of mature defensins in azurophil granules but also contain a more easily mobilized reservoir of unprocessed prodefensins in specific granules.

Overexpression of BsoBI restriction endonuclease in E. coli, purification of the recombinant BsoBI, and identification of catalytic residues of BsoBI by random mutagenesis

BsoBI is a type II restriction enzyme found in Bacillus stearothermophilus JN209 that recognizes the symmetric sequence 5′-CYCGRG-3′ (Y=C or T; R=A or G) and cleaves between the first and second base to generate a four-base 5′ extension. The cloning and sequencing of BsoBI restriction-modification system has been described by Ruan et al. [Mol. Gen. Genet. 252 (1996) 695–699]. Here we report the overexpression of BsoBI restriction endonuclease gene in E. coli by insertion of the endonuclease gene into an expression vector pRRS. The recombinant BsoBI was purified to homogeneity and its N-terminus sequence was determined. It has the same N-terminal aa sequence as the native enzyme. The constitutive expression of BsoBI from pRRS is lethal to E. coli in the absence of the cognate methylase. The bsoBIR gene was mutagenized with either hydroxylamine or by error-prone polymerase chain reaction in vitro and transferred into E. coli via plasmid vectors in the absence of the cognate methylase. Surviving transformants were selected that carry BsoBI variants which lost endonuclease activity. DNA sequencing of the mutant alleles revealed that G123, D124, D212, D246, E252 and H253 are important residues for enzymatic activity. An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-deficient variants. Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D246N, D246G and E252K can still bind DNA despite the loss of cleavage activity. Thus, residues D124, D212, D246 and E252 may be located near or within the catalytic center, and are likely involved in metal ion binding.

Outer membrane proteins of bovine Pasteurella multocida serogroup A isolates

The outer membrane proteins (OMPs) of P. multocida serotypes A3 (7 isolates), A4 (2 isolates), A3,4 and A2 (one isolate each) obtained from pneumonic cattle (10 isolates) and from one pig isolate were investigated to identify potential immunogens. SDS-PAGE of P. multocida OM isolated by SDG centrifugation of spheroplasts revealed eight major OMPs. Outer membranes isolated by sarcosyl extraction or SDG had similar protein composition on Coomassie blue-stained SDS-PA gel and on immunoblots. Two major OMPs ( M rs of 35 and 46 kDa at 100°C) demonstrated heat modifiability with apparent M rs of 30 and 34 kDa at 37°C, respectively. The N-terminal aa sequences of these heat modifiable proteins revealed homology with E. coli OmpA and Hib P1 proteins, respectively. Protease treatment of whole cells followed by western immunoblots using bovine convalescent sera identified several immunogenic, surface-exposed and conserved OMPs among the eleven P. multocida isolates examined. The whole organism SDS-PAGE profiles of the eleven P. multocida isolates differed such that six patterns were seen. These patterns could potentially be used as a typing system for P. multocida bovine isolates based on the molecular weights of whole cell proteins. The above observations have potentially important implications relative to the immunity to infection.

Cloning and sequencing of the gene encoding tannase and a structural study of the tannase subunit from Aspergillus oryzae

We cloned the Aspergillus oryzae tannase gene using three oligodeoxyribonucleotide (oligo) probes synthesized according to the tannase N-terminal and an internal amino acid (aa) sequence. The nucleotide (nt) sequence of the tannase gene was determined and compared with that of a tannase DNA complementary to RNA (cDNA) by means of reverse transcriptase PCR. The results indicated that there was no intron in the tannase gene and that it coded for 588 aa with a molecular weight of about 64 000. The tannase low-producing strain A. oryzae AO1 was transformed with the plasmid pT1 which contained the tannase gene, and tannase activities of the transformants increased in proportion to the number of copies. Tannase consisted of two kinds of subunits, linked by a disulfide bond(s) with molecular weights of about 30 000 and 33 000, respectively. We purified these subunits and determined their N-terminal aa sequences. The large and small subunits of tannase were encoded by the first and second halves, respectively. Judging from the above results, the tannase gene product is translated as a single polypeptide that is cleaved by post-translational modification into two tannase subunits linked by a disulfide bond(s). We concluded that native tannase consisted of four pairs of the two subunits, forming a hetero-octamer with a molecular weight of about 300 000.

Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase from Aspergillus fumigatus

Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.

Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase fromAspergillus fumigatus

Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus Fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.

Identification of four new prokaryotic bacterioferritins, from Helicobacter pylori, Anabaena variabilis, Bacillus subtilis and Treponema pallidum, by analysis of gene sequences

The nucleotide (nt) sequence of the Helicobacter pylori (Hp) napA gene, encoding neutrophil-activating protein A (HPNAP) was determined. Alignment of this sequence with those of known bacterioferritins (Bfr) revealed sequence homology and conservation of a 7-amino-acid (aa) motif constituting the ferroxidase (Frx) center of Bfr in the HPNAP. The N-terminal aa sequence deduced from the iron-regulated mrgC gene of Bacillus subtilis [Chen et al., J. Bacteriol. 175 (1993) 5428–5437] is highly similar to that of HPNAP and contains five Frx center aa residues. The deduced aa sequences for proteins of unknown function in Treponema pallidum [Walfield et al., Infect. Immun. 57 (1989) 633–635] and in the cyanobacterium Anabaena variabilis [Sato, GenBank accession No. JU0384 (1991)] identify these two proteins as Bfr. Although the DNA-binding protein from starved cells of Escherichia coli [Almiron et al., Genes Dev. 6 (1992) 2646–2654] is clearly a HPNAP/Bfr homologue, a significant part of its Frx center is missing. It is unlikely that the intracellular function of HPNAP is related to its ability to activate neutrophils.

Cloning and characterization of the gene encoding an extracellular alkaline serine protease from Vibrio metschnikovii strain RH530

The gene vapT, encoding VapT, one of the extracellular sodium dodecyl sulfate (SDS)-resistant alkaline serine proteases (Serp) from the Gram - Vibrio metschnikovii strain RH530 has been cloned in Escherichia coli. The recombinant E. coli produced a protease which co-migrated with VapT on gelatin polyacrylamide gels. The nucleotide (nt) sequence of the cloned vapT revealed a single open reading frame of 1641 bp encoding 547 amino acids (aa) (58 961 Da). Upon analysis of the N-terminal aa sequence, VapT was shown to be processed properly in recombinant E. coli and to consist of 428 aa (45 626 Da). The deduced aa sequence of VapT showed significant sequence homology to subtilisin Carlsberg from Bacillus licheniformis, particularly in the regions containing active site residues and calcium-binding sites. VapT had an intervening region of approx. 149 aa between the His and Ser residues of the active site, as compared with other Serp.

The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase: cloning, sequence and high-level expression in Escherichia coli

p-Nitrobenzyl esters serve as protecting groups on intermediates in the manufacture of clinically important oral β-lactam antibiotics; de-esterification of the intermediates is required for synthesis of the final product. A Bacillus subtilis PNB carboxy-esterase (PNBCE) catalyzes hydrolysis of several β-lactam antibiotic PNB esters to the corresponding free acid and PNB alcohol. This communication (i) describes cloning the pnbA gene, which encodes PNBCE, (ii) provides the nucleotide sequence of the pnbA open reading frame (ORF) and (iii) describes a method for efficiently expressing the ORF in Escherichia coli. The amino acid (aa) sequence, deduced from the nucleotide sequence of the pnbA ORF, matched an experimentally determined N-terminal aa sequence of B. subtilis PNBCE and also matched an active site sequence previously identified by biochemical analyses. Specific activity of PNBCE in crude extracts was more than 90-fold greater in recombinant E. coli, as compared to B. subtilis. This increase in expression led to more than a 500-fold improvement in the efficiency of purification of PNBCE.

Efficient secretion of biologically active mouse tumor necrosis factor alpha by Streptomyces lividans.

We have studied the production of mouse tumor necrosis factor alpha (mTNF) with Streptomyces lividans as host. mTNF cDNA was fused to the alpha-amylase-encoding gene (aml) of Streptomyces venezuelae ATCC15068 at 12 amino acids (aa) downstream from the signal-peptidase cleavage site so that the aa surrounding this processing site were conserved. S. lividans containing this construct secreted mTNF at moderately high levels (1-10 micrograms/ml) as a biologically active compound of high specific activity (1 x 10(8) units/mg protein). No unprocessed pre-protein and virtually no processed protein could be detected in the cell lysates. N-terminal aa sequence analysis indicated microheterogeneity (-3 to -6 forms) at the N-terminal site of secreted mTNF. It was demonstrated that this microheterogeneity was due to aminopeptidase activity.

Cloning, analysis and expression of the HindIII R-M-encoding genes.

The genes encoding the HindIII restriction endonuclease (R.HindIII ENase) and methyltransferase (M.HindIII MTase) from Haemophilus influenzae Rd were cloned and expressed in Escherichia coli and their nucleotide (nt) sequences were determined. The genes are transcribed in the same orientation, with the ENase-encoding gene (hindIIIR) preceding the MTase-encoding gene (hindIIIM). The two genes overlap by several nt. The ENase is predicted to be 300 amino acids (aa) in length (34,950 Da); the MTase is predicted to be 309 aa (35,550 Da). The HindIII ENase and MTase activities increased approx. 20-fold when the genes were brought under the control of an inducible lambda pL promoter. Highly purified HindIII ENase and MTase proteins were prepared and their N-terminal aa sequences determined. In H. influenzae Rd, the HindIII R-M genes are located between the holC and valS genes; they are not closely linked to the HindII R-M genes.

Alginate lyase from Klebsiella pneumoniae, subsp. aerogenes: gene cloning, sequence analysis and high-level production in Escherichia coli

The alyA gene, encoding a secreted guluronate-specific alginate lyase (Aly) from Klebsiella pneumoniae subsp. aerogenes type 25, has been cloned. DNA sequence analysis reveals two possible translation start sites for the precursor form of Aly and a long open reading frame (ORF) predicted to encode a 287-amino-acid (aa) mature form of Aly, in agreement with N-terminal aa sequence analysis of the protein. Aly has a calculated molecular mass of 31.4 kDa, in good agreement with SDS-PAGE analysis, and a calculated p I of 9.39. Comparison of the deduced aa sequence with a mannuronate-specific lyase from a marine bacterium reveals 19.3% identity and 28.8% similarity with a 9-aa conserved region close to the C terminus, probably of functional or structural significance. There is no obvious sequence similarity with pectate lyases which also catalyse a β-elimination reaction. Heterologous expression of K. pneumoniae alyA in Escherichia coli yields 10 mg of Aly per litre of culture supernatant, apparently due to non-specific release from the periplasm.

Nucleotide and deduced amino acid sequences of Rhizobium meliloti 102F34 lacZ gene: comparison with prokaryotic β-galactosidases and human β-glucuronidase

The nucleotide (nt) sequence of a 2.57-kb Sau3A. fragment carrying the Rhizobium meliloti β-galactosidase (βGal)-encoding gene ( RmlacZ) was determined. An open reading frame (ORF) of 2.26 kb was identified which encoded a 755-amino-acid (aa) polypeptide with a calculated molecular mass of 84 141 Da, in fair agreement with the value of 88 kDa determined by SDS-PAGE. The deduced N-terminal aa sequence was confirmed by direct sequencing of electro-phoretically purified R. meliloti βGal. The size of the native R. meliloti βGal was approx. 174 kDa. Similarities were found between the aa sequence of the R. meliloti βGal and those from Clostridium thermosulfurogenes EM1 and Agrobacterium radiobacter, as well as human β-glucuronidase (βGlu). Comparisons with βGal from Escherichia coli, Klebsiella pneumoniae, Lactobacillus bulgaricus and Kluyveromyces lactis found only weak similarities; however, the putative active site residues appear to be conserved. The RmlacZ sequence is flanked by two partially sequenced ORFs, which show aa sequence and organisational similarities to the previously reported lac operon in A. radiobacter.

Cloning and sequence analysis of the gene encoding the crystalline surface layer protein of Rickettsia typhi

The nucleotide sequence of the gene ( slpT) encoding the crystalline surface layer protein (SLP) of Rickettsia typhi was determined. The slpT gene consists of 4935 bp coding for a 1645-amino-acid (aa) protein containing a predicted signal peptide at the N terminus. The size of the predicted SLP exceeds the observed size (135kDa) on SDS-PAGE. The N-terminal aa sequence of the 32-kDa protein of R. typhi reported by Hackstadt et al. [Infect. Immun. 60 (1992) 159–165] was found in the C-terminal portion of the deduced aa sequence, suggesting that the product of slpT is processed into the mature SLP and the 32-kDa protein.

Cloning, sequencing, and expression of a minor protease-encoding gene from Serratia marcescens ATCC21074

The gene ( smp) encoding an extracellular protease (Smp) from Serratia marcescens ATCC21074 has been cloned and expressed in Escherichia coli HB101. The nucleotide (nt) sequence of the cloned smp gene revealed a single open reading frame of 1056 bp coding for 352 amino acids (aa) (38 479 Da). The N-terminal aa sequence of Smp excreted from the E. coli host cells revealed that mature Smp consists of 300 aa (32 515 Da). The deduced aa sequence of Smp showed high overall homology (43%) to the Erwinia carotovora metalloprotease, but low homology (15–20%) to other metallopro-teases, including the S. marcescens major metalloprotease. The location for three zinc ligands and the active site for Smp was predicted from other metalloproteases. The biochemical properties of Smp show that this enzyme is a metalloprotease whose activity is optimal at pH 8.0 and 50°C.

Sequences and expression of the porcine apolipoprotein A-I and C-III mRNAs

Apolipoprotein A-I (ApoA-I) is the principal protein component of plasma high-density lipoprotein (HDL) and an activator of lecithin:cholesterol acyltransferase. Apolipoprotein C-III (ApoC-III) exchanges between triglyceride-rich lipoproteins and HDL and inhibits the lipolysis and uptake of triglyceride-rich lipoproteins. To study the expression of these Apo-encoding genes in the developing swine, apoA-I and apoC-III cDNAs from a λgt 11 porcine liver cDNA library and apoC-III from a porcine genomic DNA library were isolated and sequenced. The predicted amino acid (aa) sequence and composition for ApoC-III matched the N-terminal aa sequence and composition of purified swine ApoCIII. Comparison among known ApoA-I and C-III aa sequences from various species revealed strict conservation of amphipathic helices. In adult pigs, the apoA-I mRNA was found predominantly in the intestine and liver, with a small amount detected in the testes. In contrast, apoC-III mRNA was found predominantly in adult liver. Developmentally, hepatic apoA-I and apoC-III mRNAs were expressed in livers of fetal, newborn, and suckling animals. Intestinal apoA-I and apoC-III mRNAs, however, were detected only in postpartum animals. Although intestinal apoA-I mRNA expression continued into the adult, intestinal apoC-III mRNA expression declined sharply after the newborn period.