Pichia Expression Research Articles

Targeted introduction of a diphtheria toxin resistant mutation into the chromosomal EF-2 locus of Pichia pastoris and expression of immunotoxin in the EF-2 mutants

In an attempt to increase the production of a diphtheria toxin (DT) based immunotoxin by Pichia pastoris, we have created DT-resistant mutants that contain a substitution of arginine for glycine at position 701 in elongation factor 2 (EF-2). To achieve this, we first cloned and characterized the EF-2 gene ( PEF1), and then made a construct pBLURA-Δ 5 ′ mutEF-2 that efficiently introduces specific mutations into the chromosomal EF-2 gene in P. pastoris by in vivo homologous recombination. pBLURA-Δ 5 ′ mutEF-2 contains a selection marker URA3 and a 5 ′ truncated form of the P. pastoris PEF1 that had been modified in vitro to carry the nucleotide mutations for the Gly 701 to Arg transition. Unlike the non-mutated strains, the EF-2 mutants are resistant to high-level intracellular expression of DT A chain that can catalyze the ADP-ribosylation. When used to express the secreted bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), the EF-2 mutant strains showed increased viability compared to the non-mutated strains. However, they did not show an advantage over the non-mutated expressing strain in the production of the immunotoxin. Western blotting analysis revealed that although the EF-2 mutants did not increase the accumulation of intact A-dmDT390-bisFv(G4S) in the culture medium, they generated larger amounts of degraded products found in both the medium and cell pellets compared to the non-mutant expressing clone. In addition, double copy expression resulted in greater amounts of intact immunotoxin being retained within cellular compartments as well as degraded products. Based on these findings, we suggest that the secretory capacity may be rate limiting for divalent immunotoxin production in P. pastoris.

Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) open reading frame 4 protein (kaposica) is a functional homolog of complement control proteins.

The genome analysis of Kaposi's sarcoma-associated herpesvirus (KSHV) has revealed the presence of an open reading frame (ORF 4) with sequence homology to complement control proteins. To assign a function to this protein, we have now expressed this ORF using the Pichia expression system and shown that the purified protein inhibited human complement-mediated lysis of erythrocytes, blocked cell surface deposition of C3b (the proteolytically activated form of C3), and served as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b (the subunits of C3 convertases). Thus, our data indicate that this KSHV inhibitor of complement activation (kaposica) provides a mechanism by which KSHV can subvert complement attack by the host.

Family 19 chitinase from rice (Oryza sativa L.): substrate-binding subsites demonstrated by kinetic and molecular modeling studies.

A family 19 chitinase (OsChia1c, class I) from rice, Oryza sativa L., and its chitin-binding domain-truncated mutant (OsChia1c deltaCBD, class II) were produced by the Pichia expression system, and the hydrolytic mechanism toward N-acetylglucosamine hexasaccharide [(GlcNAc)6] was investigated by HPLC analysis of the reaction products. The profile of the time-course of (GlcNAc)6 degradation obtained by OsChia1c was identical to that obtained by OsChia1c deltaCBD, indicating that the chitin-binding domain does not significantly participate in oligosaccharide hydrolysis. From the theoretical analysis of the reaction time-course of OsChia1c deltaCBD, the free energy changes of sugar residue binding were estimated to be -0.4, -4.7, +3.4, -0.5, -2.3, and -1.0 kcal/mol for the individual subsites of (-3), (-2), (-1), (+1), (+2), and (+3), respectively. The hexasaccharide substrate appears to bind to the enzyme through interactions at the high-affinity sites, (-2) and (+2), and the sugar residues at both ends more loosely bind to the corresponding subsites, (-3) and (+3). The docking study of (GlcNAc)6 with the modeled structure of OsChia1c deltaCBD supported the subsite structure estimated from the experimental time-course of hexasaccharide degradation. Since the class II chitinase from barley seeds was reported to possess a similar subsite structure from (-3) to (+3) and a similar free energy distribution, substrate-binding mode of plant chitinases of this class would be similar to each other.

Expression of two green fluorescent protein variants in citrate-buffered media in Pichia pastoris

The Aequorea victoria green fluorescent protein (GFP) has become one of the most frequently employed molecular reporters. A variety of mutational methods have created a large number of GFP variants with differing pH sensitivity [1], differing spectra, and optimized expression [2]. Many of the recent mutational approaches have focused on species-specific codon optimization. First established in 1980 [3], the idea that organisms do not display a random pattern of synonymous codon usage has been validated by the large amount of recent genome sequencing projects [4]. A substantial number of studies have examined the effects of varying codon bias in heterologous protein expression systems under the assumption that disparate patterns of codon bias in the transgene and the expression host will have a significant impact on the levels of recombinant protein produced. It has been suggested that the human codon-optimized enhanced green fluorescent protein (EGFP; Clontech, Palo Alto, CA) is unsuitable for expression in yeast host systems due to such preferred codon discrepancy. However, several studies have demonstrated successful expression in yeast, mainly in Saccharomyces cerevisiae [5–7]. Therefore, we compared the expression of two GFP variants, red-shifted GFP (RSGFP from pRSGFP-C1; Clontech), and EGFP, in the methylotrophic yeast Pichia pastoris. Unlike EGFP, RSGFP is not human codon-optimized; however, like EGFP, RSGFP does have an excitation wavelength shifted toward the red end of the spectrum. Both variants also exhibit greater fluorescence intensity than wildtype GFP: EGFP fluoresces 35 times greater and RSGFP fluoresces 4–6 times greater. We chose to examine the expression of both variants in citrate-buffered rather than phosphate-buffered media to determine whether either variant could serve as a fusion partner with proteins reported to exhibit inhibition in the presence of phosphate. Glucocerebrosidase (acid b-glucosidase, EC 3.2.1.45) activity, for example, has been suggested to be inhibited by phosphate ions [8]. Based on the codon bias theory, we expected to see moderate amounts of RSGFP and minimal amounts of EGFP produced. To create the RSGFP and EGFP constructs, the variant cDNAs were PCR-amplified with the following primers: 50-TAGAATTCCCGGTCGCCACCATG-30 and50-TGTTACAGGGCCCGCGGTTCAGTCGAC-30 for RSGFP; 50-AACGGTCGAATTCATGGTGAGCA AGGG-30 and 50-TATGATCTGAATTGCCGGCCGC TTTACTT-30 for EGFP. PCR amplification was performed with an initial denaturation at 94 C for 2.5min, followed by 30 cycles of 94 C for 1min, 60 C (for RSGFP) or 56 C (for EGFP) for 1.5min, and 72 C for 1.5min. Reactions were carried out with 1ll of template (pRSGFP-C1 or pEGFP-N1 vector), 5ll of 10 PCR buffer, 5ll of 25mM MgCl2, 5ll of 2.5mM dNTPs, and 4 ll forward and reverse primers in a total volume of 50 ll. These amplified cDNAs were digested with EcoR1 and ligated into the vector pPICZaA (Invitrogen, San Diego, CA) prior to electroporation into bacterial cells. Transformantswere selected by resistance toZeocin (0.1mg/ml; Invitrogen) on low-salt Luria–Bertani plates (pH 7.5). Clones were confirmed by PCR amplification and sequenced to ensure the fidelity of the PCR and cloning steps. True positive pPICZaA–RSGFP and pPICZaA– EGFP clones were isolated, purified, and linearized at the BstX1 restriction site prior to electroporation into GS115 P. pastoris cells according to the Pichia Expression Manual protocol (Invitrogen). Transformants were Analytical Biochemistry 311 (2002) 193–195

BjCHI1 from Brassica juncea displays both chitinase and agglutination activity

The proteins encoded by the Brassica juncea chitinase gene BjCHI1 and its derived genes BjCHI2 and BjCHI3 were expressed by Multi-copy Pichia expression system. The chitinase activity of FPLC purified BjCHI1, BjCHI2 and BjCHI3 were tested and the results showed that all the three proteins degraded both CM-chitin-RBV and colloidal chitin. The Km values of BjCHI1, BjCHI2 and BjCHI3 for CM-chitin-RBV were estimated as 0.799 mg/mL, 0.544 mg/mL and 0.793 mg/mL, respectively. When the colloidal chitin was used as substrate, the Km values were 0.281 mg/mL, 0.388 mg/mL and 1.643 mg/mL, respectively, indicating chitin-binding domain can increase affinity of chitinase to insoluble substrate. In the agglutination activity assay, only BjCHI1 shows activity when the protein concentration was more than 33 micrograms/mL, while BjCHI2 and BjCHI3 without agglutination activity even when the concentration was increased as high as 800 micrograms/mL. This means that the two chitin-binding domains in BjCHI1 are essential for agglutination and BjCHI1 is the first protein which shows both chitinase and agglutination activity identified so far in plants.

항균활성을 보유한 재조합 Pichia pastoris 균주의 개발

곤충에서 유래한 항균 펩티드, defensin을 항균활성이 있는 활성적인 형태로 분비하는 Pichia 균주를 개발하기 위한 일환으로서 MF<TEX>$\alpha$</TEX>1 prerpo sequence와 defensin 합성유전자를 pichia 발현 벡터에 재조합하여 형질전환하고 아미노산 histidine을 첨가하지 않은 최소 배지에서 형질전환체를 일차적으로 선별하였다. 선별한 형질전환체를 대상으로 항생제 G-418에 대한 내성과 M. luteus를 시험균으로 사용하여 생육환 저해를 통한 defensin의 세포 외 분비를 조사하여 4 균주를 선택하고 분석하였다. Southern hybridizaion을 통해 숙주의 염색체 DNA에 삽입한 defensin 유전자가 유지됨을 확인하였으며 전체 RNA를 분리하고 RT-PCR을 수행하여 defensin mRNA를 증폭하고 Southern hybridization을 실시한 결과 증폭된 밴드는 probe로 사용한 defensin 유전자에 의해 양성 신호가 나타났다. 배양시간에 따른 4 균주의 세포성장과 항균활성을 비교하기 위해 BMMY 배지에서 96시간 배양하였다. 세포성장은 모두 유사한 양상을 보였다. 세포성장은 48시간 동안 급격하게 증가한 후 그 이후로는 정지기에 도달되었다. 항균활성도 48시간까지 급격히 증가하였으며 항균활성이 가장 높은 형질전환체 균주 3는 72시간 배양 시 550 AU/<TEX>$m\ell$</TEX>을 나타내었다. To develop a yeast strain of Pichia pastoris producing an antibacterial peptide, we have attempted the expression and secretion of an insect defensin. The nucleotide sequences corresponding to mature defensin were chemically synthesized by 6 oligomers, assembled in vitro and the synthesized gene was identified by nucleotide sequencing. The prepro sequence of yeast mating factor <TEX>$\alpha$</TEX>1 and the defensin gene were recombined into a Pichia expression vector, pPIC9K. The resulting plasmid, pPIDE, was transformed into P. pastoris GSl15 and transformants selected on histidine-deficient minimal plates were tested for antibacterial activity against Micrococcus luteus. Four strains with different antibacterial activity were selected for further analysis. Southern hybridization and RT-PCR verified the defensin gene was maintained and transcribed in a host. Four strains were cultivated in YPD broth for 96 hours to compare cell growth and antibacterial activity, They showed no difference in cell growth, however, each strain showed different antibacterial activity pattern with culture time. The maximal activity was about 550 AU/ <TEX>$m\ell$</TEX>.

Cloning the gene encoding acetyl xylan esterase from Aspergillus ficuum and its expression in Pichia pastoris

Acetyl esterases are important in the complete degradation of acetylated polysaccharides, such as pectins and xylans. We isolated the gene encoding acetyl xylan esterase (AfAXE) from a genomic λ library of Aspergillus ficuum. We cloned the corresponding cDNA by RT-PCR. The Afaxe gene contained two introns, one TATAA box, and two CAAT-like boxes. The transcription initiation site was 61 bp upstream of the start codon. The deduced amino acid sequence consisted of a putative 28-amino acid leader peptide and a mature protein with an estimated molecular mass of 29.5 kDa. The nearest homolog of the cloned gene was acetyl xylan esterase of A. niger. The cloned gene was placed in a Pichia expression vector and expressed in Pichia pastoris. The culture filtrate of the transformant liberated acetyl moieties from p-nitrophenyl acetate and its activity reached 75.8 IU/ml, which was over 100-fold greater than the activity of the native enzyme expressed in A. ficuum. The cloned enzyme catalyzed the release of acetic acid from acetylated hardwood xylan, confirming that the cloned gene encoded an acetyl xylan esterase of A. ficuum. The native and recombinant acetyl xylan esterases were purified from the culture filtrates of A. ficuum and P. pastoris, respectively. Both enzymes had approximately the same optimal temperature (37 °C) and pH (7.0). The recombinant protein had greater tolerance for alkaline conditions (pH≥7.0), but was less thermostable above 55 °C.

A Family of 16-kDa Pancreatic Secretory Stress Proteins Form Highly Organized Fibrillar Structures upon Tryptic Activation

A group of 16-kDa proteins, synthesized and secreted by rat pancreatic acinar cells and composed of pancreatic stone protein (PSP/reg) and isoforms of pancreatitis-associated protein (PAP), show structural homologies, including conserved amino acid sequences, cysteine residues, and highly sensitive N-terminal trypsin cleavage sites, as well as conserved functional responses in conditions of pancreatic stress. Trypsin activation of recombinant stress proteins or counterparts contained in rat pancreatic juice (PSP/reg, PAP I and PAP III) resulted in conversion of 16-kDa soluble proteins into 14-kDa soluble isoforms (pancreatic thread protein and pancreatitis-associated thread protein, respectively) that rapidly polymerize into insoluble sedimenting structures. Activated thread proteins show long lived resistance to a wide spectrum of proteases contained in pancreatic juice, including serine proteases and metalloproteinases. In contrast, PAP II, following activation with trypsin or pancreatic juice, does not form insoluble structures and is rapidly digested by pancreatic proteases. Scanning and transmission electron microscopy indicate that activated thread proteins polymerize into highly organized fibrillar structures with helical configurations. Through bundling, branching, and extension processes, these fibrillar structures form dense matrices that span large topological surfaces. These findings suggest that PSP/reg and PAP I and III isoforms consist of a family of highly regulated soluble secretory stress proteins, which, upon trypsin activation, convert into a family of insoluble helical thread proteins. Dense extracellular matrices, composed of helical thread proteins organized into higher ordered matrix structures, may serve physiological functions within luminal compartments in the exocrine pancreas.

Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris.

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.

Recombinant Expression of α‐Bungarotoxin in Pichia pastoris Facilitates Identification of Mutant Toxins Engineered to Recognize Neuronal Nicotinic Acetylcholine Receptors

A snake venom-derived alpha-neurotoxin, alpha-bungarotoxin (alphaBgtx), is the classic competitive antagonist of nicotinic acetylcholine receptors (nAChRs). The very high specificity and essentially irreversible binding of alphaBgtx to various nAChRs make alphaBgtx the prime candidate for studying the molecular determinants of specificity for nAChR-ligand interactions. To facilitate site-directed mutagenesis of alphaBgtx for functional analysis, we have developed a recombinant expression system for alphaBgtx using the methylotropic yeast Pichia pastoris. A synthetic gene coding for alphaBgtx was subcloned into an expression vector that directs secretion of the recombinant alphaBgtx (rBgtx) when stably integrated into the yeast genome. Expression of rBgtx was induced by growth of yeast cultures with methanol as the sole carbon source. The activity of the rBgtx in the cell-free medium was measured by competition with 1251-Bgtx for binding to Torpedo nAChR-enriched membranes. The rBgtx, purified to homogeneity by standard HPLC, has the correct predicted amino terminal sequence and molecular mass. Its circular dichroism spectrum is very similar to that of authentic venom-derived alphaBgtx, and the biological activity of the rBgtx is identical to that of authentic alphaBgtx. We have used the Pichia expression system to study a double point mutation of alphaBgtx, rBgtx-K38P/L42Q, that has a high affinity for alpha3beta2 neuronal nAChRs. This is the first demonstration of engineering an alpha-neurotoxin to recognize non-alpha7 neuronal nicotinic receptors.

Expression of mouse α-amylase gene in methylotrophic yeastPichia pastoris

The expression of the mouse α -amylase gene in the methylotrophic yeast, P. pas- toris was investigated. The mouse α -amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested with SalI or BglII, and was introduced into P. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested with SalI or BglII into the HIS4 locus (38 of Mut + clone) or into the AOX1 locus (15 of Mut S clone). Southern blot was carried out in 11 transformants, which showed that the mouse α -amylase gene was integrated into the Pichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α -amylase activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α -amylase gene is compared with that in recombinant Saccharomyces cerevisiae harboring a plasmid encoding the same mouse α -amylase gene, the specific enzyme activity is eight fold higher than that of the recombinant S. cerevisiae.

Cloning and Expression in Pichia Pastoris of a Genetically Engineered Single Chain Antibody Against the Rat Transferrin Receptor

The present investigation describes the construction of a genetically engineered single chain antibody (scFv) against the rat transferrin receptor (OX26), and demonstrates that this scFv antibody can be fully processed and expressed as a soluble secreted molecule in the methylotrophic yeast Pichia pastoris. Restriction endonuclease sites located at both 5′- and 3′-flanking regions of OX26 coding region in the prokaryote pOPE-OX26 vector were engineered to incorporate yeast compatible restriction endonuclease sites (i.e. EcoRI and Smal or AvrII). The modified OX26 cDNA was subcloned into the Pichia expression vectors pPIC9 and pHIL-S1. An OX26 scFv high producer clone [GS115 His+ Mut+ (pPIC-OX26 SacI)] was isolated and used for large-scale production and characterization. Because the engineered scFv contains both a c-myc tag and a (His)5 tail, the OX26 scFv was purified to homogeneity by immobilized metal affinity chromatography. The identity of the OX26 scFv was confirmed by Western blot analyses with both and c-myc and anti poly-His antibodies. Minor immunoreactive bands corresponding to hyperglycosylated and partially processed α-factor leader prosequence were also detected in the purified OX26 scFv, and these contaminants were markedly reduced when the expression of the OX26 scFv was performed in minimal methanol medium buffered with phosphate at pH = 7. The present investigation suggests that this expression system may be useful for the production of anti-receptor single chain antibodies that can be used as brain drug delivery vectors.

Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris.

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.

Human placental alkaline phosphatase: expression in Pichia pastoris, purification and characterization of the enzyme.

The soluble form of human placental alkaline phosphatase (PLAP) was expressed in the methylotrophic yeast Pichia pastoris and the expression product was purified and characterized. Yeast-derived PLAP (yPLAP) was secreted into the medium to the level of 2 mg/liter. yPLAP displayed kinetic properties similar to those reported earlier for the membrane-bound PLAP. Purified yPLAP had specific activity of 774 U/mg and appeared in two subunit sizes, ca. 62 and 65 kDa. This difference was due to heterogenous N-glycosylation. Purified yPLAP appeared as multiple forms in isoelectric focusing in pI range of 4.2 to 5.2. The expression system is discussed in comparison to previously reported expression systems.

Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase.

Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human alpha subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI. Coexpression with human type III procollagen unexpectedly increased the assembly level 10-fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4-hydroxyproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47-like protein. The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4-hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high-level production of various recombinant collagens for numerous scientific and medical purposes.

Riboflavin 5′-Hydroxymethyl Oxidation: MOLECULAR CLONING, EXPRESSION, AND GLYCOPROTEIN NATURE OF THE 5′-ALDEHYDE-FORMING ENZYME FROM SCHIZOPHYLLUM COMMUNE

Vitamin B2-aldehyde-forming enzyme catalyzes oxidation of the 5'-hydroxymethyl of riboflavin to the formyl group. We have purified the enzyme from the culture media of Schizophyllum commune (ATCC 38719) by modifying the procedure of Tachibana and Oka (Tachibana, S., and Oka, M. (1981) J. Biol. Chem. 256, 6682-6685) for cell-free extract. By SDS-polyacrylamine gel electrophoresis, the enzyme appears to be 78 kDa. The enzyme has a blocked amino terminus, so fragments were obtained by cleaving the purified enzyme with lysyl endopeptidase. Selected peptides were sequenced from their amino termini. We have isolated a full-length cDNA clone using a DNA hybridization probe amplified by polymerase chain reaction with two degenerate oligonucleotide primers, the design of which was based on one of the partial amino acid sequences. From the cDNA clone, it is evident that the enzyme has a Ser/Thr-rich fragment near the COOH-terminal Asp. The enzyme was determined to be a glycoprotein; however, O-deglucosylation only slightly affects activity. Computer searches showed that the B2-aldehyde-forming enzyme has little homology with other proteins, but domain motifs may reflect N-myristoylation of a dehydrogenase with a signature similar to 4Fe-4S ferredoxins. The enzyme cDNA was subcloned into a Pichia expression vector pPIC9K to produce a recombinant protein which exhibited B2-aldehyde-forming enzyme activity.

Expression inPichia pastorisand Purification ofAspergillus awamoriGlucoamylase Catalytic Domain

In this paper we report the expression inPichia pastoris,purification, and characterization of theAspergillus awamoriglucoamylase catalytical domain (GAc).Pichia pastorisproduced GAc to the level of 0.4 g per liter medium. This production level is about the same level as that gained for recombinant GA fromAspergillusand about 100-fold more than previously achieved bySaccharomyces cerevisiae.The GAc expressed inPichia pastoriswas purified by two independent chromatographic methods employing ion exchange or affinity chromatography to apparent homogeneity. The purified protein has a molecular weight of about 75,000 and specific activity of 78 units per milligram protein. The propeptide present in the glucoamylase N terminus was found to be removed correctly byP. pastoris.Glucoamylase produced byP. pastorisis N- and O-glycosylated, with 23% carbohydrate content. The N-linked oligosaccharides appear to be larger than in invertase, another glycoprotein heterologously expressed inP. pastoris.O-glycosides (studied to our knowledge for the first time inP. pastorisin this report) contribute about half of the total carbohydrate content in GAc. Purified GAc appears as multiple bands on isoelectric focusing with pIvalues around 3.5, a value that is little higher than that for GAc produced inS. cerevisiae.GAc could be used as a versatile tool in studying protein expression inP. pastoris:as an affinity handle for other secreted proteins produced inP. pastoris,as a reporter gene when studying gene expression, and as a model protein in studying protein secretion and processing inP. pastoris.

High level secretion of single-chain antibody in Pichia expression system

Single chain Fv-170 (scFv-170) was constructed and expressed in the methylotrophic yeast Pichia pastoris. Recombinant scFv-170 was highly expressed in an aF-signal based expression vector and sufficiently secreted from the cells in protein-free medium. Further, the scFv-170 maintained the intrinsic binding activity and exhibited the same binding pattern as that of MAb 170H.82, as measured in competitive radioimmunoassay (RIA) experiments.

Cloning of thePenicillium minioluteum gene encoding dextranase and its expression inPichia pastoris

Cloning of thePenicillium minioluteum gene encoding dextranase and its expression inPichia pastoris

Human Alanyl-tRNA Synthetase: Conservation in Evolution of Catalytic Core and Microhelix Recognition

The class II Escherichia coli and human alanyl-tRNA synthetases cross-acylate their respective tRNAs and require, for aminoacylation, an acceptor helix G3:U70 base pair that is conserved in evolution. We report here the primary structure and expression in the yeast Pichia of an active human alanyl-tRNA synthetase. The N-terminal 498 amino acids of the 968-residue polypeptide have substantial (41%) identity with the E. coli protein. A closely related region encompasses the class-defining domain of the E. coli enzyme and includes the part needed for recognition of the acceptor helix. As a result, previously reported mutagenesis, modeling, domain organization, and biochemical characterization on the E. coli protein appear valid as a template for the human protein. In particular, we show that both the E. coli enzyme and the human enzyme purified from Pichia aminoacylate 9-base pair RNA duplexes whose sequences are based on the acceptor stems of either E. coli or human alanine tRNAs. In contrast, the sequences of the two enzymes completely diverge in an internal portion of the C-terminal half that is essential for tetramer formation by the E. coli enzyme, but that is dispensable for microhelix aminoacylation. This divergence correlates with the expressed human enzyme behaving as a monomer. Thus, the region of close sequence similarity may be a consequence of strong selective pressure to conserve the acceptor helix G3:U70 base pair as an RNA signal for alanine.