Expression, Characterization, and Purification of Recombinant Porcine Lactoferrin in Pichia pastoris

Wang, Sue-Hong; Yang, Tien-Shuh; Lin, Shiang-Ming; Tsai, Ming-Shiun; Wu, Shinn-Chih; Mao, Simon J.T.

doi:10.1006/prep.2001.1607

Cited by 44 publications

(35 citation statements)

References 37 publications

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“…As shown in Figure 5, based on the peak heights of yeast broth and standard hLF (1.0 mg·mL −1 ), it is estimated that the concentration of expression hLF was about 1.2 mg·mL −1 , which is significantly higher than those reported by Liang and Richardson [12] and Ying et al [28]. In addition, the secretion of recombinant LF can increase by adjusting the initial pH of the culture from 6 to 7 and by the addition of ferric ions at 100 μmol·L −1 to the medium [25].…”

Section: Optimization Of Hlf Expressionmentioning

confidence: 73%

“…The Pichia pastoris system has now been used successfully to express a large number of intracellular and secreted proteins. Extremely high levels have been obtained for some biologically active molecules [6], and LF has been shown to be expressed in P. pastoris with the highest yield of 115 mg·L −1 [17,18,25,28]. In this paper, the effect of the ratio of glycerol and methanol on the expression of hLF and the growth of yeast cells were determined by dot blotting and OD600.…”

Section: Optimization Of Hlf Expressionmentioning

confidence: 99%

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High-level expression and production of human lactoferrin in Pichia pastoris

Jiang

Chen

Jia

et al. 2008

Dairy Sci. Technol.

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-Lactoferrin (LF) is a multifunctional iron-binding glycoprotein which is found in high concentrations in milk. Recombinant human LF (rhLF) may provide health benefits. In the present study, we report an optimization of the production system of recombinant human lactoferrin that has enabled the production of recombinant protein at levels up to 1200 mg·L −1 . The hLF was expressed in the methylotrophic yeast Pichia pastoris using the pPIC9K vector. The expression level of recombinant hLF (rhLF) was improved significantly by mixed methanol/glycerol feeding at the ratio of 4:1 during the induction phase of high cell-density fermentation. A yield of approximately 1200 mg·L −1 was obtained in fed-batch fermentation with this system, which was much higher than the reported values for other systems (1 mg·L −1 to 115 mg·L −1 ). The rhLF was purified via ionexchange chromatography using SP Sepharose TM Fast Flow and has a similar molecular mass of 80 kg·mol −1 to native hLF. The level of glycosylation of the recombinant protein is similar to that of the native protein.human lactoferrin / Pichia pastoris / high cell-density fermentation / glycosylationRésumé -Haut niveau d'expression et de production de lactoferrine humaine par Pichia pastoris. La lactoferrine (LF) est une glycoprotéine multifonctionnelle liant le fer, qui se trouve en concentration élevée dans le lait. La lactoferrine humaine recombinante (rhLF) peut être bénéfique pour la santé. Dans cette étude, nous présentons une optimisation du système de production de rhLF permettant d'obtenir des niveaux de production élevés. La lactoferrine humaine était exprimée dans la levure méthylotrophe Pichia pastoris à l'aide du vecteur pPIC9K. Le niveau d'expression de rhLF était amélioré significativement lorsque le milieu était alimenté d'un mélange méthanol/glycérol dans un ratio de 4:1 pendant la phase d'induction de la fermentation à haute densité cellulaire.

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Section: Optimization Of Hlf Expressionmentioning

confidence: 73%

Section: Optimization Of Hlf Expressionmentioning

confidence: 99%

High-level expression and production of human lactoferrin in Pichia pastoris

Jiang

Chen

Jia

et al. 2008

Dairy Sci. Technol.

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“…Each lobe binds one Fe 3+ ion. In some cases the expression levels of hLF have been influenced by supplementation of FeCl 3 [36]. In order to identify the optimal concentration of FeCl 3 , the induction medium was supplemented with FeCl 3 in the range of 0.05-2.0 mM final concentration.…”

Section: Optimization Of Recombinant Human Lactoferrin Expression In mentioning

confidence: 99%

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

et al. 2008

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Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.

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“…[15][16][17] Previous reports have demonstrated that functional proteins of N-half molecule (N-lobe) of human serum transferrin and ovotransferrin can be produced in microorganisms (Pichia pastoris and Escherichia coli, respectively). [18][19][20] Although the P. pastoris expression system of the whole molecule of porcine lactoferrin and equine lactoferrin have been developed recently, 21,22) the microorganism production system of whole molecule of Fe 3þ -transporter proteins, such as serum transferrin and ovotransferrin has not been available.…”

mentioning

confidence: 99%