Purification of Recombinant Glycoproteins from Pichia pastoris Culture Supernatants

Insects express arthro-series glycosphingolipids, which contain an α1,4-linked GalNAc residue. To determine the genetic basis for this linkage, we cloned a cDNA (CG17223) from Drosophila melanogaster encoding a protein with homology to mammalian α1,4-glycosyltransferases and expressed it in the yeast Pichia pastoris. Culture supernatants from the transformed yeast were found to display a novel UDP-GalNAc:GalNAcβ1,4GlcNAcβ1-R α-N-acetylgalactosaminyltransferase activity when using either a glycolipid, p-nitrophenylglycoside or an N-glycan carrying one or two terminal β-N-acetylgalactosamine residues. NMR and MS in combination with glycosidase digestion and methylation analysis indicate that the cloned cDNA encodes an α1,4-N-acetylgalactosaminyltransferase. We hypothesize that this enzyme and its orthologues in other insects are required for the biosynthesis of the N5a and subsequent members of the arthro-series of glycolipids as well as of N-glycan receptors for Bacillus thuringiensis crystal toxin Cry1Ac.

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Enrichment and identification of Δ 9 -Tetrahydrocannabinolic acid synthase from Pichia pastoris culture supernatants

Data in Brief ◽

10.1016/j.dib.2015.07.033 ◽

2015 ◽

Vol 4 ◽

pp. 641-649 ◽

Cited By ~ 2

Author(s):

Kerstin Lange ◽

Ansgar Poetsch ◽

Andreas Schmid ◽

Mattijs K. Julsing

Keyword(s):

Pichia Pastoris ◽

Culture Supernatants

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Characterization of N-Linked Glycosylation on Recombinant Glycoproteins Produced in Pichia pastoris Using ESI-MS and MALDI-TOF

Glycomics ◽

10.1007/978-1-59745-022-5_16 ◽

2008 ◽

pp. 213-223 ◽

Cited By ~ 4

Author(s):

Bing Gong ◽

Michael Cukan ◽

Richard Fisher ◽

Huijuan Li ◽

Terrance A. Stadheim ◽

...

Keyword(s):

Pichia Pastoris ◽

Esi Ms ◽

Maldi Tof ◽

Recombinant Glycoproteins

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Metabolic engineering of CHO cells to prepare glycoproteins

Emerging Topics in Life Sciences ◽

10.1042/etls20180056 ◽

2018 ◽

Vol 2 (3) ◽

pp. 433-442 ◽

Cited By ~ 1

Author(s):

Qiong Wang ◽

Michael J. Betenbaugh

Keyword(s):

Metabolic Engineering ◽

Cho Cells ◽

Genetic Manipulation ◽

Chinese Hamster ◽

Post Translational Modification ◽

Effector Functions ◽

Recombinant Glycoproteins ◽

Production Platform ◽

Chemical Inhibitors ◽

Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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Differential Detection of Single-Chain and Two-Chain Urokinase-Type Plasminogen Activator by a New Immunoadsorbent-Amidolytic Assay (IAA)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661692 ◽

1986 ◽

Vol 56 (03) ◽

pp. 407-410 ◽

Cited By ~ 10

Author(s):

Angelo Corti ◽

Maria Luisa Nolli ◽

Giovanni Cassani

Keyword(s):

Plasminogen Activator ◽

Carcinoma Cell ◽

Differential Detection ◽

Single Chain ◽

Response Curves ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Before And After ◽

Culture Supernatants ◽

Human Epidermoid Carcinoma

SummaryA new immunoadsorbent-amidolytic assay (IAA) for the specific differential detection of two-chain urokinase-type plasminogen activator (tcu-PA) and its single-chain precursor (scu-PA) in cell culture supernatants has been developed. The assay combines the selectivity of immunoassays with the specificity of enzyme activity assays exploiting both the antigenic and enzymatic properties of the two proteins. tcu-PA and scu-PA are selectively immunoadsorbed on the wells of a microtiterplate coated with the monoclonal antibody 5B4 and tested for enzymatic activity before and after activation by plasmin treatment. Both proteins are determined with similar efficiency since overlapping dose-response curves were obtained in the range between 12.5-200 ng/ml. The assay has been used to determine tcu-PA and scu-PA in A431 human epidermoid carcinoma cell supernatants. The analytical recoveries for tcu-PA and scu-PA added to A431 cell supernatants were 95.2% and 96.9% respectively. The intra- and inter-assay variations (CV) were 5.5% and 9.0% for tcu-PA and 9.7% and 9.8% for scu-PA respectively.

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Optimization of fermentation conditions for the production of recombinant hpdgf-bb in Pichia pastoris

TAP CHI SINH HOC ◽

10.15625/0866-7160/v37n1se.6119 ◽

2015 ◽

Vol 37 (1se) ◽

Author(s):

Duong Long Duy ◽

Pham Minh Vu ◽

Nguyen Tri Nhan ◽

Tran Linh Thuoc ◽

Dang Thi Phuong Thao

Keyword(s):

Pichia Pastoris ◽

Fermentation Conditions ◽

Optimization Of Fermentation

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Culture of Human A375 Melanoma Cells in the Presence of Fibronectin Causes Expression of MMP-9 and Activation of MMP-2 in Culture Supernatants

Journal of Environmental Pathology Toxicology and Oncology ◽

10.1615/jenvironpatholtoxicoloncol.v27.i2.60 ◽

2008 ◽

Vol 27 (2) ◽

pp. 135-145 ◽

Cited By ~ 6

Author(s):

Aniruddha Banerji ◽

Shamik Das ◽

Amitava Chatterjee

Keyword(s):

Melanoma Cells ◽

Culture Supernatants ◽

A375 Melanoma

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Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

International Journal of Pharmaceutical Quality Assurance ◽

10.25258/ijpqa.10.1.22 ◽

2019 ◽

Vol 10 (01) ◽

Author(s):

Rafid A. Abdulkareem

Keyword(s):

Pichia Pastoris ◽

Human Insulin ◽

Expression Vector ◽

Expression System ◽

Insulin Gene ◽

Cloning And Expression ◽

Pcr Analysis ◽

Major Band ◽

Human Insulin Gene ◽

Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

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