Expression of an anti-CD33 single-chain antibody by Pichia pastoris

CD33 is a cell surface glycoprotein expressed on cells of myelomonocytic lineage, leukaemic cells, but not haematopoietic stem cells. By virtue of its expression pattern, CD33 has become a popular target for new immunotherapeutic approaches to treat acute myeloid leukaemia. The methylotrophic yeast Pichia pastoris strain KM71H was used to produce an anti-CD33 single chain variable fragment (scFv), with the intention of conjugation to a radioisotope, for therapeutic use. To direct secreted expression of the anti-CD33-scFv the alpha-mating factor secretory signal sequence (alpha-MF) was used, with constructs containing a complete (CS) and incomplete (INCS) cleavage site to accommodate the potential outcomes of dibasic endopeptidase, Kex2, and dipeptidyl amino peptidase, Ste13, processing. The anti-CD33-scFv was expressed in BMMY cultures using both constructs, with a final yield of 48 mg/l (CS) and 11 mg/l (INCS). N-terminal sequencing showed that the CS-scFv had not been cleaved by Ste13, leaving amino acids EAEA at the N-terminus. The INCS-scFv construct produced a mixture of 50% authentic scFv and 50% with 11 amino acids from the alpha-MF remaining at the N-terminus. Despite the aberrations in alpha-MF processing, the anti-CD33-scFv's produced from both constructs were found to be functional. Flow cytometry and Biacore analysis demonstrated binding to target antigen CD33 on the surface of human leukaemic cell line HL-60, and to recombinant soluble CD33 respectively

Production of a soluble and functional recombinant apolipoproteinD in the Pichia pastoris expression system

ApolipoproteinD (ApoD) is a human glycoprotein from the lipocalin family. ApoD contains a conserved central motif of an 8-stranded antiparallel β-sheet, which forms a beta-barrel that can be used for transport and storage of diverse hydrophobic ligands. Due to hydrophobic nature of ApoD, it has been difficult to generate a recombinant version of this protein. In the present work, we aimed at the production of ApoD in the robust Pichia pastoris expression system. To this end, the ApoD gene sequence was synthesized and subcloned for expression in the yeast host cells. Following integration of the ApoD gene into the yeast genomic region using homologous recombination, the ApoD recombinant protein was induced using methanol, reaching its maximum induction at 96 h. Having purified the ApoD recombinant protein by affinity chromatography, we measured the dissociation constant (KD) using its natural ligands: progesterone and arachidonic acid. Our results provide a viable solution to the production of recombinant ApoD protein in lieu of previous obstacles in generating soluble and functional ApoD protein. © 2016 Elsevier Inc. All rights reserved

Optimized expression of the Starmerella bombicola lactone esterase in Pichia pastoris through temperature adaptation, codon-optimization and co-expression with HAC1

The Starmerella bombicola lactone esterase (SBLE) is a novel enzyme that, in vivo, catalyzes the intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment. In fact, this is an unusual reaction given the unfavorable conditions for dehydration. This characteristic strongly contributes to the potential of SBLE to become a 'green' tool in industrial applications. Indeed, lactonization occurs normally in organic solvents, an application for which microbial lipases are increasingly used as biocatalysts. Previously, we described the production of recombinant SBLE (rSBLE) in Pichia pastoris (syn. Komagataella phaffii). However, expression was not optimal to delve deeper into the enzyme's potential for industrial application. In the current study, we explored codon-optimization of the SBLE gene and we optimized the rSBLE expression protocol. Temperature reduction had the biggest impact followed by codon-optimization and co-expression of the HAC1 transcription factor. Combining these approaches, we achieved a 32-fold improvement of the yield during rSBLE production (from 0.75 mg/l to 24 mg/L culture) accompanied with a strong reduction of contaminants after affinity purification

Cloning and Extracellular Expression of Glargine in Pichia pastoris

Patients with diabetes mellitus increase significantly every year. The increasing number of people with diabetes mellitus results in increased insulin requirements. There are two types of insulin used for diabetes mellitus treatment: human insulin and insulin analogues. Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpaha has been used to produce human insulin and insulin analogues. Pichia pastoris can produce glargine in large quantities, and the insulin protein produced will be secreted outside the cell to facilitate the purification process. The advantage of glargine has a long working time of up to 24 hours. Hence, glargine is more effective because patients with diabetes receive glargine injections only once daily. The research started with cloning the glargine gene, transforming pPICZαA-G plasmid into Pichia pastoris, and testing glargine production. 20 recombinant Pichia pastoris colonies were selected and regenerated. Eight recombinant Pichia pastoris colonies were tested for glargine production, and six colonies were detected producing glargine by electrophoresis SDS-PAGE gel stained with Coomassie blue. This study aims to produce glargine using Pichia pastoris as an expression system capable of producing glargine extracellularly, thus simplifying the purification process

Mutational analysis of the carbohydrate binding activity of the tobacco lectin

At present the three-dimensional structure of the tobacco lectin, further referred to as Nictaba, and its carbohydrate-binding site are unresolved. In this paper, we propose a three-dimensional model for the Nictaba domain based on the homology between Nictaba and the carbohydrate-binding module 22 of Clostridium thermocellum Xyn10B. The suggested model nicely fits with results from circular dichroism experiments, indicating that Nictaba consists mainly of beta-sheet. In addition, the previously identified nuclear localization signal is located at the top of the protein as a part of a protruding loop. Judging from this model and sequence alignments with closely related proteins, conserved glutamic acid and tryptophan residues in the Nictaba sequence were selected for mutational analysis. The mutant DNA sequences as well as the original Nictaba sequence have been expressed in Pichia pastoris and the recombinant proteins were purified from the culture medium. Subsequently, the recombinant proteins were characterized and their carbohydrate binding properties analyzed with glycan array technology. It was shown that mutation of glutamic acid residues in the C-terminal half of the protein did not alter the carbohydrate-binding activity of the lectin. In contrast, mutation of tryptophan residues in the N-terminal half of the Nictaba domain resulted in a complete loss of carbohydrate binding activity. These results suggest that tryptophan residues play an important role in the carbohydrate binding site of Nictaba

Efficient display of active lipase LipB52 with a Pichia pastoris cell surface display system and comparison with the LipB52 displayed on Saccharomyces cerevisiae cell surface

<p>Abstract</p> <p>Background</p> <p>For industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.</p> <p>Results</p> <p>Two <it>Pichia pastoris </it>cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the α-factor secretion signal sequence were constructed respectively. The lipase gene <it>lipB52 </it>fused with the <it>FLO </it>gene was successfully transformed into <it>Pichia pastoris </it>KM71. The lipase LipB52 was expressed under the control of the <it>AOX1 </it>promoter and displayed on <it>Pichia pastoris </it>KM71 cell surface with the two <it>Pichia pastoris </it>cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the <it>Pichia pastoris </it>cell surface exhibited activity toward <it>p</it>-nitrophenol ester with carbon chain length ranging from C₁₀to C₁₈, and the optimum substrate was <it>p</it>-nitrophenol-caprate (C₁₀), which was consistent with it displayed on the <it>Saccharomyces cerevisiae </it>EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on <it>Pichia pastoris </it>KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40°C at pH8.0, they retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.</p> <p>Conclusion</p> <p>The LipB52 displayed on the <it>Pichia pastoris </it>cell surface exhibited better stability than the lipase LipB52 displayed on <it>Saccharomyces cerevisiae </it>cell surface. The displayed lipases exhibited similar transesterification activity. But the <it>Pichia pastoris </it>dry cell weight per liter (DCW/L) ferment culture was about 5 times than <it>Saccharomyces cerevisiae</it>, the lipase displayed on <it>Pichia pastoris </it>are more suitable for whole-cell biocatalysts than that displayed on <it>Saccharomyces cerevisiae </it>cell surface.</p

Development Of High Cell Density Recombinant Pichia Pastoris Culture For Human Growth Hormone Production

Methylotrophic yeast, Pichia pastoris has been used as an excellent host to produce more than 400 recombinant heterologous proteins of academic and industrial interests. Pichia pastoris telah digunakan sebagai perumah yang unggul untuk menghasilkan lebih daripada 400 jenis protein heterologus kegunaan bidang akademik dan industri

Pichia pastoris Fep1 is a [2Fe-2S] protein with a Zn finger that displays an unusual oxygen-dependent role in cluster binding

Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys(2)-Cys(2) type zinc fingers and a set of four conserved cysteines arranged in a Cys-X-5-Cys-X-8-Cys-X-2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster

PENGARUH VARIASI KONSENTRASI METANOL DAN LAMA INDUKSI TERHADAP EKSPRESI PROINSULIN OLEH Pichia pastoris SECARA INTRASELULER

The Effects of Variation in Methanol Concentration and Induction Time on Intracellular Proinsulin Expression by Pichia pastoris ABSTRACTDiabetes is a metabolic disorder characterized by hyperglycemia. There were 215 million diabetic patients in 2014 and the number is expected to rise in 2040. Generally, insulin is used to treat diabetic patients. Insulin production by recombinant technology has been done, though still inefficient, by using E. coli and S. cerevisiae expression system. Another alternative expression system is methylotrophic yeast Pichia pastoris. In this research, proinsulin has been expressed by P. pastoris intracellularly. P. pastoris strains used in this research were X33, GS115, and KM71H. All recombinant strains were MutS. Best cultivation media was BMGY. Proinsulin expression was observed at 25°C. Pichia pastoris strain that expressed proinsulin best was GS115-PI. It was supported by PCR in which the strain GS115-PI gave 504 bp-sized bands. Based on proinsulin formation time, the final methanol concentration of 0.5% in 72 hours was found to be the best treatment.Keywords: BMGY, methanol, phenotype, Pichia pastoris, proinsulin ABSTRAKDiabetes melitus merupakan kelainan yang ditandai dengan hiperglikemia. Penderita diabetes pada tahun 2014 di dunia mencapai 215 juta dan diperkirakan akan meningkat pada tahun 2040. Umumnya penderita diabetes diberi pengobatan insulin sehingga menunjukkan akan ada peningkatan kebutuhan insulin. Produksi insulin dengan teknologi DNA rekombinan telah dilakukan dengan menggunakan sistem ekspresi E. coli dan S. cerevisiae namun masih belum efisien. Sistem alternatif lain adalah ragi metilotropik Pichia pastoris. Dalam penelitian ini dilakukan ekspresi proinsulin dari P. pastoris secara intraseluler. Galur P. pastoris yang digunakan dalam penelitian ini adalah X33, GS115, dan KM71H. Semua galur rekombinan adalah MutS. Media tumbuh terbaik adalah BMGY. Ekspresi proinsulin terlihat pada suhu 25°C. Hasil PCR menunjukkan bahwa galur GS115-PI yang dapat menghasilkan pita amplikon berukuran 504 bp. Hasil PCR ini dibuktikan oleh hasil seleksi galur yang menunjukkan bahwa galur GS115-PI dapat mengekspresi proinsulin dibandingkan galur lainnya. Berdasarkan kecepatan pembentukan pita protein proinsulin, variasi konsentrasi akhir metanol 0,5% dengan lama induksi 72 jam merupakan perlakuan terbaik.Kata Kunci: BMGY, fenotipe, metanol, Pichia pastoris, proinsuli