Elsevier

Journal of Biotechnology

Volume 99, Issue 1, 9 October 2002, Pages 51-62
Journal of Biotechnology

High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris

https://doi.org/10.1016/S0168-1656(02)00157-8Get rights and content

Abstract

The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. In order to provide proteins for the ‘protein structure factory’, a structural genomics initiative, we are working on the high-throughput expression of human proteins. Therefore, cDNAs are cloned for intracellular expression. The resulting fusion proteins carry affinity tags (6*HIS and StrepII, respectively) at the N- and C-terminus for the immunological detection and chromatographic purification of full-length proteins. Expression is controlled by the tightly regulated and highly inducible alcoholoxidase 1 (AOX1) promoter. We have developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression. The screening procedure is based on a culture volume of 2 ml in a 24-well format. Lysis of the cells occurs via a chemical lysis without mechanical disruption. Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones which produce heterologous protein with a yield of 5 mg l−1 culture volume or higher.

Introduction

After publishing the draft sequence of the human genome (International Human Genome Sequencing Consortium, 2001), efforts are on the way to understand the function of coded proteins on the genomic scale. One step towards this goal is to elucidate the three-dimensional structure of the coded proteins (Abbott, 2000). Therefore, the structural genomics initiative ‘Protein Structure Factory’ was set up to solve protein structures in a high-throughput, automated manner (Heinemann et al., 2000; www.proteinstrukturfabrik.de). Target proteins are heterologously expressed in different hosts. Pichia pastoris was established as an alternative host for the ‘Protein Structure Factory’ in addition to Escherichia coli and Saccharomyces cerevisiae in order to provide an expression system for those proteins that are expressed in insufficient quantities or not at all in the other hosts.

The methylotrophic yeast P. pastoris is a powerful tool for the heterologous expression of proteins (reviewed by Cereghino and Cregg, 2000, Faber et al., 1995). This system combines several advantages of prokaryotic and eukaryotic expression systems. High yields of recombinant protein are reported (Cereghino and Cregg, 2000). The techniques needed for molecular genetic manipulation are similar to those well established for S. cerevisiae. P. pastoris can be easily grown to high cell densities using defined minimal media and is able to introduce eukaryotic posttranslational modifications. Distribution and chain length of N-linked oligosaccharides are different to those of S. cerevisiae, namely the chain length is significantly shorter, making it an interesting alternative for the extracellular expression of human proteins (Grinna and Tchopp, 1989). Another advantage of the P. pastoris expression system for structural analysis purposes is the possibility of 13C labeling of expressed proteins for nuclear magnetic resonance analyses. Feeding 13C labeled methanol provides a cost-effective possibility to achieve incorporation levels of up to 98% (Laroche et al., 1994).

The proteins of interest to our project are expressed under the control of the alcoholoxidase 1 (AOX1) promoter. This promoter is strongly induced by methanol, which also serves as the main carbon source while inducing the expression (Ellis et al., 1985) and is repressed by most other carbon sources (Tschopp et al., 1987). Typically, expression clones of P. pastoris are grown in excess of a repressing carbon source, e.g. glucose or glycerol, followed by inducing protein expression by the addition of methanol after depletion of the repressing carbon source (Cregg et al., 1993). Methanol concentration in the culture must be kept within a relatively narrow range. Accumulation of methanol leads to cytotoxic effects (Guarna et al., 1997) and the induction of the promoter is highest when the methanol level is kept to growth limiting rates (Couderc and Baratti, 1980).

Mixed-feeding strategies where methanol and glycerol are fed simultaneously during the induction phase have been applied to increase the yield of the heterologous protein in both MutS strains (Loewen et al., 1997) and Mut+ strains (McGrew et al., 1997). At least in some cases feeding of glycerol at levels below the repressing concentration leads to an increased biomass formation and higher yield of recombinant protein (McGrew et al., 1997).

In this paper we describe a screening system for high-throughput characterization of P. pastoris expression clones transformed with different cDNAs. The fusion of proteins with affinity tags allows immunological detection of expression. Furthermore, the purification of the proteins can be faciliated under native conditions. Polyhistidine fusion proteins are purified via immobilized metal affinity chromatography (IMAC; Bruel et al., 2000), StrepII tagged proteins are isolated using their affinity to a StrepTactin matrix (Voss and Skerra, 1997). A procedure amendable to automation was established in order to screen transformants for protein expression. Detection limits of this procedure were estimated.

Section snippets

Strains and media

E. coli XL-1 blue (Stratagene, USA) was used for cloning.

All experiments were performed with the haploid P. pastoris strain GS115 (his4; Invitrogen, USA). Transformed cells were selected on YNB Plates w/o amino acids (Becton, Dickinson and Company, USA) supplied with 2% glucose.

Expression experiments were carried out in different liquid media. YNB w/o amino acids and double concentrated YNB (Becton, Dickinson and Company, USA) according to manufacturers instructions were used as commercially

Construction of the expression vector

The expression vector pPICHS is based on the plasmid pPIC3.5 (Invitrogen, USA). Modifications comprise the introduction of a translation initiation sequence (AAAATGTCT) followed by a sequence coding for six histidine residues. The vector furthermore codes for a C-terminal StrepII tag (IBA, Germany) followed by a stop codon (see Fig. 1). The inserts were cloned via BamHI/NotI. cDNAs were amplified by PCR. Primers were designed to introduce a BamHI site at the 5′ end and a NotI site at the 3′

Acknowledgements

We thank Natalia Bolotina for amplifying the cDNAs. Heiko Kraemer provided excellent technical assistance. We thank Roslin Bensmann for help with the English. This work was supported by the Bundesministerium für Bildung und Forschung through the Leitprojekt ‘Proteinstrukturfabrik’.

References (22)

  • L.S. Grinna et al.

    Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris

    Yeast

    (1989)
  • Cited by (112)

    • Bovine immunisation with a recombinant peptide derived from synthetic SBm7462® (Bm86 epitope construct) immunogen for Rhipicephalus microplus control

      2020, Ticks and Tick-borne Diseases
      Citation Excerpt :

      These transformed yeasts were cultured in selective medium MD without histidine (1.34 % YNB, 4 × 10−5% biotin, 2% dextrose, 1.5 % bacteriologic agar). Posteriorly, the grown yeasts were isolated, replicated for five generations in the same MD medium with ampicillin (Roche) and the stable insertion of the expression cassette was checked by colony PCR using the AOX1-promoter and terminator specific primers (Boettner et al., 2002). Finally, the clones were selected using the colony blot technique according to the method proposed by Maniak (1989) using polyclonal IgG anti-SBm7462® produced in rabbits.

    • Enhancing the efficiency of recombinant hepatitis B surface antigen production in Pichia pastoris by employing continuous fermentation

      2019, Biochemical Engineering Journal
      Citation Excerpt :

      These steps include glycerol feed in batch and fed-batch mode, to reach high cell densities, methanol adaptation and subsequent fed-batch fermentation for rHBsAg expression [28–31]. To improve the efficiency of rHBsAg production in the fermentation process, different approaches have been suggested [6,12,32–34]. These approaches include using different vector constructs, optimization growth medium composition and applying different strategies for glycerol and methanol feeding [21,25,27,35].

    • Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability

      2017, Enzyme and Microbial Technology
      Citation Excerpt :

      Pichia pastoris, an effective host for the production of both secreted and intracellular heterologous proteins, can help overcome the disadvantages of the Escherichia coli system, since it is capable of disulfide bond formation, proteolytic maturation, N-and O-linked glycosylations, and other post-translational modifications [10]. At present, high-throughput screening of the Pichia pastoris expression system is mainly accomplished using phenotypic selection in microtiter well plates [11–15]. However, screening using microtiter well plates not only results in a heavy workload and requires expensive instruments for colony operation, but also takes a relatively long time.

    • A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection

      2016, Vaccine
      Citation Excerpt :

      Different Zeocin concentrations in the range 100-1000 mg/ml were tested, and clones with resistance to higher antibiotic concentrations were selected. The insertion of the DNA fragment was checked by colony PCR in selected Zeocin-resistant colonies [27]. PCR positive clones were cultured in minimal methanol histidine plates.

    View all citing articles on Scopus
    View full text