Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals

Human interferon-alpha 2b (IFN-α2b) was cloned and expressed in Pichia pastoris under the control of alcohol oxidase promoter (AOX1) using three different secretion signals. Native secretion signal of IFN-α2b, Saccharomyces cerevisiae MF-α factor prepro sequence and a mutated α prepro sequence without the Glu-Ala (EAEA) repeats were used separately for directing the secretion of IFN-α2b into the culture medium of P. pastoris. The native secretion signal of IFN-α2b did not secrete protein into the culture medium of P. pastoris. The α prepro sequence without the EAEA repeats directed the secretion of maximum amount of IFN-α2b (200 mg/l) into the culture medium, with the same amino acid sequence as that of the native IFN-α2b secreted by human lymphocytes. The full α prepro sequence, having both the protease cleavage sites for KEX2 and STE13 gene products, also secreted an equivalent amount of IFN-α2b into the culture medium. However, two interferon bands with similar molecular masses were observed, when full α prepro sequence was used for the secretion of IFN-α2b. The difference in the molecular masses of the two bands was found to arise due to the difference in the molecular masses of the N-terminal fragment, and the inefficient processing of secretion signal.     

Synthesis, secretion and processing of alpha-factor-interferon fusion proteins in yeast.

A gene fusion consisting of 960 base pairs of 5'-flanking region of the yeast MF alpha 1 gene, 257 base pairs coding for alpha-factor prepro sequence, and a modified human IFN-alpha 1 gene was constructed. MAT alpha cells containing the chimeric gene synthesized and secreted active IFN-alpha 1 into the growth medium. The secreted interferon molecules contained the last 4 amino acids of alpha-factor prepro sequence and the amino acids encoded by the DNA modifications introduced at the beginning of IFN-alpha 1 gene. DNA sequences coding for these amino acids were removed by oligonucleotide-directed in vitro mutagenesis. Yeast cells transformed with expression plasmids containing the altered junction synthesized and secreted human IFN-alpha 1 with the natural NH2-terminus.     

Sequence of an intestinal cDNA encoding human motilin precursor

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.     

A full length cDNA clone for the alkaline protease from Aspergillus oryzae: structural analysis and expression in Saccharomyces cerevisiae

Summary We have cloned and determined the nucleotide sequence of a cDNA fragment for the entire coding region of the alkaline protease (Alp) from a filamentous ascomycete Aspergillus oryzae. According to the deduced amino acid sequence, Alp has a putative prepro region of 121 amino acids preceding the mature region, which consists of 282 amino acids. A consensus sequence of a signal peptide consiting of 21 amino acids is found at the N-terminus of the prepro region. The primary structure of the mature region shares extensive homology (29%–44%) with those of subtilisin families, and the three residues (Asp 32, His 64 and Ser 221 in subtilisin BPN) composing the active site are preserved. The entire cDNA, coding for prepro Alp, when introduced into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium, with its N-terminus and specific activity identical to native Aspergillus Alp.     

Identification and structure-activity relationship of an antimicrobial peptide of the palustrin-2 family isolated from the skin of the endangered frog Odorrana ishikawae

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.     

A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger

Abstract The secreted yield of hen egg-white lysozyme (HEWL) from the filamentous fungus Aspergillus niger was increased 10–20-fold by constructing a novel gene fusion. The cDNA sequence encoding mature HEWL was fused in frame to part of the native A. niger gene encoding glucoamylase ( gla A), separated by a proteolytic cleavage site for in vivo processing. Using this construct, peak secreted HEWL yields of 1 g/l were obtained in A. niger shake flask cultures compared to about 50 mg/l when using an expression cassette lacking any gla A coding sequence. The portion of gla A used in the gene fusion encoded the first 498 amino acids of glucoamylase (G498) and comprised its secretion signal, the catalytic domain and most of the O-glycosylated linker region which, in the entire glucoamylase molecule, spatially separates and links the catalytic and starch-binding domains.     

Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ^{5, 8, 11, 14}) and eicosapentaenoic acid (EPA; 20:5 Δ^{5, 8, 11, 14, 17}). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy. Yun-Tao Li and Mao-Teng Li contributed equally to this work.     

Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris

We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l^–1 when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.     

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.     

Green flavoprotein from P. leiognathi: Purification,characterization and identification as the product of the lux G(N) gene

A green flavoprotein (GFP) was isolated and purified to homogeneity from Photobacterium leiognathi, strain 208. GFP is a homodimer of molecular weight 54000 and contains two molecules of an unusual flavin per molecule of protein. Various biochemical characteristics including isoelectric point, trypsin and chymotrypsin degradation, SDS and temperature influence on subunit dissociation and the dissociation of the flavin chromophore, were investigated. The sequence of 23 N-terminal amino acids was determined and found to be concurrent with the N-terminal amino acids was determined and found to be concurrent with the N-terminal amino acid sequence encoded by the lux G (N) gene of P. leiognathi. This fact suggests that GFP is a structural component of the Photobacterium luminescence system.     

Expression of Methyl Parathion Hydrolase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

In the Pichia pastoris expression system, increasing the copy number of the expression cassette often has the effect of increasing the amount of protein expressed. To improve the expression level of methyl parathion hydrolase (MPH), we constructed two integration vectors with four and eight direct repeats of the expression cassette using an in vitro multimerization approach. After two successive integrations, at least 12 copies of the MPH expression cassette were integrated into the P. pastoris chromosome. Under shake-flask conditions, over 55 mg active MPH/l was secreted into the medium by the multicopy clones. The extracellular enzyme activity was about 10-fold higher for the multicopy clones than for clones containing a single copy of the gene. Further investigations revealed that the multicopy MPH expression cassette could remain stably integrated and functional over five generations. Note that the expression vector pRF constructed in our study can be not only used to construct multiple copies of the expression cassette in vitro, but also integrated into the P. pastoris genome without introducing any antibiotic resistance gene, which is desirable for production of biotherapeutic proteins.     

Complete amino acid sequence of the N-terminal extension of calf skin type III procollagen.

The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain.     

Molecular Cloning and Expression of Yak (Bos grunniens) Lactoferrin cDNA in Pichia pastoris

cDNA encoding lactoferrin from yak was isolated by RT-PCR and then sequenced. The cloned cDNA (2127 bp) encodes a 709 amino acid precursor molecule of yak lactoferrin with a signal peptide of 19 amino acids. The yak lactoferrin cDNA was expressed in Pichia pastoris. The recombinant protein, purified by Ni-NTA affinity column, had a molecular weight of 76 kDa and reacted with an antibody raised against native bovine lactoferrin. The iron-binding behavior and antimicrobial activity of the purified protein indicated that it was correctly folded and functional.     

Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the ascomycete Melanocarpus albomyces

The lac1 gene encoding an extracellular laccase was isolated from the thermophilic fungus Melanocarpus albomyces. This gene has five introns, and it encodes a protein consisting of 623 amino acids. The deduced amino acid sequence of the laccase was shown to have high homology with laccases from other ascomycetes. In addition to removal of a putative 22-amino-acid signal sequence and a 28-residue propeptide, maturation of the translation product of lac1 was shown to involve cleavage of a C-terminal 14-amino-acid extension. M. albomyces lac1 cDNA was expressed in Saccharomyces cerevisiae under the inducible GAL1 promoter. Extremely low production was obtained with the expression construct containing laccase cDNA with its own signal and propeptide sequences. The activity levels were significantly improved by replacing these sequences with the prepro sequence of the S. cerevisiae alpha-factor gene. The role of the C-terminal extension in laccase production in S. cerevisiae was also studied. Laccase production was increased sixfold with the modified cDNA that had a stop codon after the native processing site at the C terminus.     

Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone.

Two genes, MF alpha 1 and MF alpha 2, coding for the alpha-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization of synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide alpha-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MF alpha 1 gene contains 4 copies of the coding sequence for the alpha-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first alpha-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala and 83 additional amino acids. MF alpha 2 gene contains coding sequences for two copies of the alpha-factor that differ from each other and from alpha-factor encoded by MF alpha 1 gene by a Gln leads to Asn and a Lys leads to Arg substitution. The first copy of the alpha-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the alpha-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.     

Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris

Exendin-4 is a naturally occurring 39 amino acid peptide that is useful for the control of Type 2 diabetes. Recombinant Exendin-4, with an extra glycine at the carboxy-terminus (Exdgly), was expressed in the methylotropic yeast Pichia pastoris. A high proportion of the Exdgly molecules secreted into medium were found to be clipped, lacking the first two amino acids (His–Gly) from the N-terminus. Disruption of the P. pastoris homolog of the Saccharomyces cerevisiae dipeptidyl aminopeptidase (STE13) gene in Pichia genome resulted in a clone that expressed N-terminally intact Exdgly. Elimination of N-terminal clipping enhanced the yield and simplified the purification of Exdgly from P. pastoris culture supernatant.     

Molecular Structure and Properties of Lectin from Tomato Fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro₄ motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Recombinant expression of human cathelicidin (hCAP18/LL-37) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.     

Expression of a functional barley sucrose-fructan 6-fructosyltransferase in the methylotrophic yeast Pichia pastoris

The cDNA encoding sucrose-fructan 6-fructosyltransferase (6-SFT) from barley (Hordeum vulgare) has been expressed in the methylotrophic yeast Pichia pastoris, using a translational fusion into vector pPICZαC, containing the N-terminal signal sequence of Saccharomyces cerevisiae α-factor to allow entry into the secretory pathway. Transformed Pichia produced and secreted a functional 6-SFT which had characteristics similar to the barley enzyme, but had a pronounced additional 1-SST activity when incubated with sucrose.