SILAC compatible strain of Pichia pastoris for expression of isotopically labeled protein standards and quantitative proteomics

Abstract

The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the P. pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications and has advanced the organism as a model system for the study of peroxisome biogenesis and methanol metabolism. Here we report the development of a P. pastoris arg-/lys- auxotrophic strain compatible with SILAC (stable isotope labeling by amino acids in cell culture) proteomic studies, which is capable of generating large quantities of isotopically labeled protein for mass spectrometry-based biomarker measurements. We demonstrate the utility of this strain to produce high purity human serum albumin uniformly labeled with isotopically heavy arginine and lysine. In addition, we demonstrate the first quantitative proteomic analysis of methanol metabolism in P. pastoris, reporting new evidence for a malate-aspartate NADH shuttle mechanism in the organism. This strain will be a useful model organism for the study of metabolism and peroxisome generation.

Figure 1

Generation of P. pastoris arg- lys- auxotroph. (A) ARG4 and LYS2 knockout vectors. The NTC (nourseothricin acetyltransferase) and HPT (hygromycin phosphotransferase) disruption vectors were used to knockout arginosuccinate lyase (ARG4) and α-aminoadipate reductase (LYS2) by homologous recombination. (B) PCR-based confirmation of genetic disruption: At left, rk88/rk6 primers amplified a 2488 amplicon generated in the arg- knockout vs. a 1222 amplicon in the ‘wild type’ BG08. At right, rk166/rk167 primers amplify a 1952 amplicon generated in lys- knockout vs. no amplicon in the wild type.

Figure 2

Heavy HSA expression and MS analysis. (A) Recombinant HSA is secreted into the culture medium at a yield of 50 μg/ml detectable by Coomasie stained SDS-PAGE. A minor 43kD degradation product of the full length HSA (67kD) is also evident, consistent with previous reports of protein cleavage in P. pastoris . The absence of other secreted proteins in the media is apparent in both methanol induced and uninduced samples. (B) MS-analysis of trypsinized HSA covers 55% of the protein sequence. Of 31 non-redundant peptides, 22 peptides show quantifiable L/H ratios by MS. (C) Sample chromatogram overlay for HSA peptide LVTDLDK with the heavy peptide AUC (gray) vs. light peptide AUC (slate).

Figure 3

P. pastoris protein profiles at 48 hours growth on methanol supplemented with heavy arginine and lysine. (A) Normalized plot of heavy:light ratios for 197 quantified proteins. Proteins up-regulated 8-fold or greater are highlighted (slate bars) and tabulated at right. The majority of up-regulated proteins are involved in methanol metabolism. (B) Schematic of methanol metabolism in P. pastoris. 1) alcohol oxidase, 2) catalase, 3) S-hydroxymethyl dehydrogenase, 4) S-formyl glutathione hydrolase, 5) formate dehydrogenase, 6) dihydroxyacetone synthase, 7) dihydroxyacetone kinase, 8) fructose 1,6-bisphosphate aldolase, 9) fructose 1,6-bisphosphatase, 10) glutathione reductase, 11) glutathione peroxidase.

Figure 4

Time course of individual protein ratios on methanol media. Precipitous up-regulation of methanol metabolic enzymes corresponds with the gradual down-regulation of glycolytic enzymes involved in glycerol metabolism. AOX1 (alcohol oxidase 1), CTA (catalase), DAS1 (dihydroxyacetone synthase 1); DAK (dihydroxyacetone kinase), FBA (fructose 1,6-bisphosphate aldolase); FLD (S-hydroxymethyl dehydrogenase), FGH (S-formyl glutathione hydrolase), FDH (formate dehydrogenase); GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK (phosphoglycerate kinase), PGM (phosphoglycerate mutase), ENO (enolase), PK (pyruvate kinase).