The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P. pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems. The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein. Included in these considerations are the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers. The impact of the alcohol oxidase (AOX) pathways in terms of the mut+ and muts phenotypes, intracellular expression and folding pathways is examined. The roles of pre–pro signal sequences such as the alpha mating factor (α-MF) and the Glu–Ala repeats at the kex2p cleavage site on the processing of the protein translate(s) have also been considered. Protocols for the generation of protein variants and mutants for screening for orphan cognate binding partners and the use of experimental platforms addressing the molecular recognition behaviour of recombinant proteins such as the extracellular domains of transmembrane receptors with their physiological ligands are also described. Finally, the palindromic patterns of glycosylation that can occur with these expression systems, in terms of the role and location of the sequon in the primary structure, the number of mannose units and the types of oligosaccharides incorporated as Asn- or O-linkages and their impact on the thermostability and immunogenicity of the recombinant protein are considered. Procedures to prevent glycosylation through manipulation of cell culture conditions or via enzymatic and site-directed mutagenesis methods are also discussed. Copyright © 2004 John Wiley & Sons, Ltd.

Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production

In the year 2003 there was a 17% increase in the number of publications citing work performed using optical biosensor technology compared with the previous year. We collated the 962 total papers for 2003, identified the geographical regions where the work was performed, highlighted the instrument types on which it was carried out, and segregated the papers by biological system. In this overview, we spotlight 13 papers that should be on everyone's 'must read' list for 2003 and provide examples of how to identify and interpret high-quality biosensor data. Although we still find that the literature is replete with poorly performed experiments, over-interpreted results and a general lack of understanding of data analysis, we are optimistic that these shortcomings will be addressed as biosensor technology continues to mature.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jmr.753

Survey of the year 2003 commercial optical biosensor literature

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.

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Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: A review

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut(+) expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(H(c))], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consumption rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (mu) determined from the model was 0.08 h(-1) at a methanol concentration of 3.65 g/L, while the actual maximum mu was 0.0709 h(-1). The maximum specific methanol consumption rate was 0.0682 g/g WCW/h. From the model, growth can be defined as either methanol-limited or methanol-inhibited and is delineated at a methanol concentration of 3.65 g/L. Under inhibited conditions, the observed biomass yield (Y(X/MeOH)) was lower and the maintenance coefficient (m(MeOH)) was higher than compared to limited methanol conditions. The Y(X/MeOH) decreased and m(MeOH) increased with increasing methanol concentration under methanol-inhibited conditions. BoNT/A(H(c)) content in cells (alpha) under inhibited growth was lower than that under limited growth, and decreased with increasing methanol concentration. A maximum alpha of 1.72 mg/g WCW was achieved at a mu of 0.0267 h(-1) and induction time of 12 h.

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Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A.

Effect of methanol concentration on the production of human β2-glycoprotein I domain V by a recombinant Pichia pastoris: A simple system for the control of methanol concentration using a semiconductor gas sensor

Measurement of association rate constant of antibody-antigen interaction in solution based on enzyme-linked immunosorbent assay.

A kinetic model for a biopanning process considering antigen desorption and effective antigen concentration on a solid phase.

To isolate phages displaying a practical and useful antibody with a high k on value and/or a low k off value from phage display antibody libraries, we developed a rational strategy based on a kinetic model. In the model, the recovery of a phage displaying an antibody after a round of biopanning is expressed as a function of five parameters, the apparent association rate constant of the phage antibody to the immobilized antigen ( k on ′), the apparent dissociation rate constant of the phage antibody from the immobilized antigen ( k off ′), the effective antigen concentration ( C ), the time for the binding process ( t b ) and the time for the washing process ( t w ). An optimum set of operating parameters ( C , t b and t w ) for isolating phages displaying an antibody with a high k on value was designed based on the model. Three rounds of biopanning were carried out under the designed conditions, against a phage library in which the hypervariable regions of an original antibody were randomized. All isolated phages displayed an antibody with a higher k on value and one displayed an antibody with a 30-fold greater k on value than that of the original antibody. Experimental conditions which improve the efficiency of conventional off-rate selections are also described.

Guoqiang Zhuang

Papers

Effect of methanol concentration on the production of human β2-glycoprotein I domain V by a recombinant Pichia pastoris: A simple system for the control of methanol concentration using a semiconductor gas sensor

Measurement of association rate constant of antibody-antigen interaction in solution based on enzyme-linked immunosorbent assay.

A kinetic model for a biopanning process considering antigen desorption and effective antigen concentration on a solid phase.

A practical kinetic model for efficient isolation of useful antibodies from phage display libraries

Analysis and Optimization of Biopanning Process of Phage Display Libraries