Search keywords: 'Pichia pastoris transferrin' Search Date 1/22/07

Some abstracts of interest:

*Sheng Wu Gong Cheng Xue Bao. 2005 Sep;21(5):804-8. [Expression of fusion protein of parathyroid hormone and transferrin N-terminal half-molecule in Pichia pastoris] [Article in Chinese]

Zhang H, Li XJ, Wang DJ, Chen J, Li YY, Li YL, Shen MS, Fang HQ, Chen HP.

Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.

The fused gene (PTH-TFN) of parathyroid hormone (PTH) gene and transferring N-terminal half-molecule (TFN) gene was amplified by multiple PCR and inserted into pPIC9 vector. The recombinant plasmid pPIC9-PTH-TFN was transformed into Pichia pastoris GS115 by PEG. After methanol induction, the target protein was expressed in fermentation supernatant at high level. The fused protein PTH-TFN with purity being higher than 95% was finally obtained after purification through two-step chromatography: SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. Western blot analysis and adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis and the ability to bind Fe3+ in the Fe3+ saturation study as the recombinant TFN did indicating that TFN could be used as the transcellar carrier of PTH.

PMID: 16285525 [PubMed - in process]

• Biosci Biotechnol Biochem. 2004 Feb;68(2):376-83. Structural and functional characterization of ovotransferrin produced by Pichia pastoris.

Mizutani K, Okamoto I, Fujita K, Yamamoto K, Hirose M.

The Graduate School of Agriculture, Kyoto University, Japan.

Ovotransferrin is an egg white protein with complex disulfide and bilobal structures, which is derived from the same gene as chicken serum transferrin. We demonstrate here the structural and functional characteristics of bilobal ovotransferrin, produced at a high level using Pichia pastoris expression system. The recombinant protein was secreted into the medium, and the secretion signal peptide was processed correctly. The secretion level was almost 100 mg/l culture and the yield after purification by two-step anion exchange chromatography was 57 mg/l. The CD spectrum and fluorescence spectra indicate the correct folding of the recombinant protein. The analyses for the Fe3+ binding ability by urea-PAGE and visible absorption spectrum revealed that two Fe3+ sites exist in a recombinant ovotransferrin molecule as in the egg white protein. Endoglycosidases, such as endo-beta-N-acetylglucosaminidase H (Endo-H), peptide:N-glycosidase F (PNGaseF? ), and endo-beta-N-acetylglucosaminidase from Mucor hiemalis, showed differential activities for the native Fe3+-loaded, native Fe3+-free, and denatured forms of recombinant ovotransferrin; only the first enzyme displayed the cleavage ability for all the ovotransferrin forms. The results from the enzyme specificity and from the molecular weight difference for the intact and deglycosylated proteins were consistent with the view that recombinant ovotransferrin have one N-linked carbohydrate chain which mainly consists of two GlcNac? and 10 mannoses.

PMID: 14981301 [PubMed - indexed for MEDLINE]

*Biochemistry. 1999 Feb 23;38(8):2535-41. X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32.

Bewley MC, Tam BM, Grewal J, He S, Shewry S, Murphy ME, Mason AB, Woodworth RC, Baker EN, MacGillivray? RT.

Institute of Molecular Biosciences, College of Sciences, Massey University, Palmerston North, New Zealand.

The ferric form of the N-lobe of human serum transferrin (Fe(III)-hTF/2N) has been expressed at high levels in Pichia pastoris. The Fe(III)-hTF/2N was crystallized in the space group P41212, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 A resolution. This represents only the second P. pastoris-derived protein structure determined to date, and allows the comparison of the structures of recombinant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with serum-derived transferrin. The polypeptide folding pattern is essentially identical in all of the three proteins. Mass spectroscopic analyses of P. pastoris- hTF/2N and proteolytically derived fragments revealed glycosylation of Ser-32 with a single hexose. This represents the first localization of an O-linked glycan in a P. pastoris-derived protein. Because of its distance from the iron-binding site, glycosylation of Ser-32 should not affect the iron-binding properties of hTF/2N expressed in P. pastoris, making this an excellent expression system for the production of hTF/2N.

PMID: 10029548 [PubMed - indexed for MEDLINE]

• Biochemistry. 1998 Sep 29;37(39):13696-703. Iron release is reduced by mutations of lysines 206 and 296 in recombinant N-terminal half-transferrin.

Steinlein LM, Ligman CM, Kessler S, Ikeda RA.

Department of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400, USA.

Human serum transferrin consists of two iron-binding lobes connected by a short peptide linker. While the high homology and structural similarity between the two halves of the molecule would suggest similar characteristics, it has been shown that the pH-dependent rate of release of iron from the N-terminal lobe is quite different from that of its C-terminal counterpart. This suggests that the N-lobe of human serum transferrin has a specific, pH-dependent, molecular mechanism for releasing iron. Sacchettini and co-workers using structural information have hypothesized that two lysines in the N-terminal lobe of ovotransferrin create a dilysine interaction and suggest that this is the trigger for pH-dependent iron release. To investigate this hypothesis, we used a Pichia pastoris expression system to produce large amounts of wild-type nTf, the single point mutants, nTfK206A (Lys 206 to alanine) and nTfK296A (Lys 296 to alanine), and the double mutant, nTfK206/296A. The purified recombinant proteins were then used to measure rates of iron release to pyrophosphate. It was found that the rate of iron release from all three mutant proteins at pH 5.7 (the pH at which nTf would normally release iron) was too slow to measure. Only when the pH was reduced to 5.0 could the rates of iron release from the mutant proteins be reliably determined. Although this precludes a direct comparison to wild-type nTf (the rate of iron release from nTf at pH 5.0 is too fast to measure), it implicates lysines 206 and 296 in the pH-dependent release of iron from nTf.

PMID: 9753457 [PubMed - indexed for MEDLINE]

*Biochemistry. 1997 Apr 8;36(14):4327-36. Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family.

Wuebbens MW, Roush ED, Decastro CM, Fierke CA.

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family. We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library. Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column. Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved. However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin. pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding. In addition, the antigenic determinants of pICA and the transferrins are not identical. These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.

PMID: 9100029 [PubMed - indexed for MEDLINE]

• Protein Expr Purif. 1996 Aug;8(1):119-25. Production and isolation of the recombinant N-lobe of human serum transferrin from the methylotrophic yeast Pichia pastoris.

Mason AB, Woodworth RC, Oliver RW, Green BN, Lin LN, Brandts JF, Tam BM, Maxwell A, MacGillivray? RT.

Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont, 05405, USA.

The N-lobe of human serum transferrin has been expressed in the methylotrophic yeast Pichia pastoris by placing the hTF/2N cDNA under the control of the methanol-inducible alcohol oxidase promoter. Following induction with methanol, the N-lobe was efficiently secreted into a basal salt medium in shake flasks at a level of 150-240 mg/liter. As judged by mobility on SDS-PAGE, immunoreactivity with two domain-specific monoclonal antibodies, and both thermal stability and spectral properties (indictative of correct folding and ability to bind iron), the recombinant N-lobe produced by the yeast cells appears to be identical to that produced in a mammalian expression system. Electrospray-mass spectrometry and a third domain specific antibody, however, show that approximately 80% of the protein from the yeast cells contains one or two hexose residues.

PMID: 8812842 [PubMed - indexed for MEDLINE]

*Protein Expr Purif. 1995 Oct;6(5):619-24. Production and purification of N-terminal half-transferrin in Pichia pastoris.

Steinlein LM, Graf TN, Ikeda RA.

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400, USA.

Human serum transferrin, the major iron transport protein in humans, is a monomeric glycoprotein that is composed of two homologous domains; the N-terminal domain is formed by amino acids 1-331 and the C-terminal domain is formed by amino acids 338-679. Each domain is capable of binding one iron atom concomittantly with a carbonate anion; however, the two homologous iron binding sites are not chemically equivalent. The cDNA sequence coding for the N-terminal domain has been cloned and overexpressed in the methylotrophic yeast, Pichia pastoris. The transformants secrete a protein of approximately 38 kDa (the size expected for N-terminal half-transferrin), its N-terminal sequence agrees with the predicted sequence, and the protein reacts with anti-human serum transferrin antibodies. The purified protein appears to be properly folded and can bind iron as demonstrated by its spectral properties and urea-PAGE mobility. It is estimated that N-terminal half-transferrin represents approximately 90% of all protein secreted into the culture medium and that it is expressed at levels exceeding 50 mg/l. This study demonstrates that N-terminal half-transferrin can easily be expressed in the simple host system, Pichia pastoris, and that the purified protein is capable of reversibly binding iron.

PMID: 8535154 [PubMed - indexed for MEDLINE]

-- DaniPershouse - 22 Jan 2007