Strategy for kinase expression in P. pastoris
We applied the same workflow for expression and purification for all target kinases. Constructs were prepared using the Gibson assembly method and transformed into yeast for expression tests, as detailed in Materials and Methods. Each target kinase was tagged with the small cleavable purification tags Strep II (WSHPQFEK) or His10 (Figure 1, Table 1). Transformants in P. pastoris typically produced different amounts of recombinant protein. We therefore screened multiple (4-10) single clones of the transformants, and the clone exhibiting the highest expression level was selected for scale-up. (Supplementary Figure 1)
The expression conditions such as induction time, temperature and methanol concentration (for protein induction) were optimized for the highest protein production. For the first day, a temperature of 30 °C was chosen to optimize yeast growth and biomass accumulation in buffered glycerol-complex medium (BMGY). After 24 hours, protein production was induced by adding 0.5% methanol and growing in buffered methanol-complex medium (BMMY) for 48 hrs at 28 °C to maximize protein production. Cells were then harvested, stored at -40 °C or processed at the same day as detailed in the materials section. Following purification on three serially connected, 5 ml Strap-Trap columns, proteins were either subjected to a second purification step when needed or directly characterized by polyacrylamide gel electrophoresis (PAGE), western blotting, and kinase activity assay. Protein purity was estimated from SDS-PAGE and enzymatic activity was measured using the ADP-Glo Max in vitro kinase enzymatic assay kit (Promega). Activity was compared to commercially available kinases with reported activity measured using the same substrates.
Expression and purification of MKK3b
MKK3 (isoform b) is a cytosolic kinase widely involved in cellular signaling. This dual specificity kinase is part of the RAS/MAPK pathway, which plays a key role in regulating proliferation, differentiation and survival (14). The active enzyme was expressed with a small Strep II tag in P. pastoris with good purity as judged by SDS-PAGE (Figure 2). After elution from a StrepTrap column, higher molecular weight aggregates were detected by SDS-PAGE. To purify the monomeric form, the eluent was pooled, diluted in buffer (50 mM Tris HCl pH8.5, 150 mM NaCl, 5% glycerol and 1 mM TCEP) and injected onto a gel filtration column (HiLoad 16/60 Superdex 200). The fractions containing the monomeric soluble protein were collected and concentrated and the identity was confirmed by Western blotting with anti-Strep antibody (Figure 2F). The yield was 0.5 mg/L culture and the activity was comparable to the commercially available MKK3 using the same reported substrate, inactive p38 (SignalChem, Table 1). The enzyme was previously expressed with a bulky MBP tag and His tag in E. coli; the inactive protein required co-expression with its upstream activator MEKK-C to obtain 25% yield of active enzyme. (14,15) Two other reports expressed a constitutively active mutant in BES with no reported yield. (16,17)
Expression and purification of full-length AurKA
The mitotic kinase AurKA has recently gained interest as a target in several malignancies. (18) Typically, AurKA is recombinantly expressed as the catalytic domain, but we were interested in the potential functions of the disordered N-terminal domain. We therefore expressed and purified strep-tagged, full-length AurKA in P. pastoris using a single purification step (StrepTrap). Purified protein expressed as monomer, 95% pure on SDS-PAGE and active. The identity was confirmed by Western blotting with anti-AurKA antibody and the yield was 1 mg/L culture (Table 1, Figure 3). We did not try to activate AurKA by phosphorylation or by addition of activating protein partner Tpx2. Nevertheless, activity was approximately 25-30% of commercially available kinase using the same substrate, MBP (18.7 nmol/mg/min vs 62-73 nmol/mg/min; SignalChem).
Expression and purification of CyclinD2 (CycD2), CDK 4 and 6
Cyclin-dependent kinases are involved in cell cycle regulation and are considered important targets in human cancers. The kinases are almost always co-expressed using BES with a cyclin to improve the kinase structural stability, folding and activity. (19) N-terminal Strep-tagged CDK4 and CDK6 were expressed in P. pastoris after co-transformation with N-terminal His6-CycD2 to obtain cyclin-CDK complexes. Complex formation was confirmed on small scale of 100 ml culture by dual-tag tandem affinity chromatography; the complex co-eluted from both StrepTrap and HisTrap columns as confirmed by SDS-PAGE and Western blotting with specific antibodies (Figure 4). Large scale, 1 L culture for protein expression was processed on a StrepTrap column with higher purity and yield for the CDK6-CycD2 complex than the CDK4-CycD2 complex (0.30 mg/L, 95% vs 0.14 mg/L, 50%, respectively; Figure 5, Table 1). Further purification by HisTrap for CDK4-CycD2 complex did not improve the purity. Both CDK4 and 6 showed slightly improved activity compared to the commercially available versions, which were co-expressed with cyclin D1 in SF9 insect cells using the same substrate, Rb protein (773 - 928). Activity was 14.4 nmol/mg/min vs 12 nmol/mg/min for CDK4/cyclin and 17.1 nmol/mg/min vs 8-9.3 nmol/mg/min for CDK6/cyclin (SignalChem).
Attempted expression of the single proteins CDK4, CDK6 and CycD2 in P. pastoris with several tags was not as successful. CDK6 did not express at all despite several rounds of transformation and expression tests. CycD2 expressed but was insoluble, while CDK4 expressed in a soluble form but with very low purity and yield (Supplementary Table 1). These results agree with most published reports describing the difficulties in expressing the individual proteins. (19)
We noted that the poorly expressing CDK4/6 and CycD2 were acidic proteins, with pI values of 6.08, 5.74, and 5.25, respectively (Table 1), while MKK3 was closer to neutral (pI = 6.28), and full-length AurKA was basic (pI = 9.25). The N- terminal domain of AurKA contributed to the overall basicity of the protein, and we hypothesized that addition of a basic domain would improve expression levels. To test this idea, we added a highly basic 110 amino acid N-terminal domain from the secreted kinase VLK (Molecular weight = 11.4 KDa, pI = 11.7) to the kinase domains of CDK6 and HER2 (see below) and monitored the effect on expression. In contrast to CDK6-Strep, which did not express in P. pastoris in the absence of CycD2, His10-VLK-CDK6-Strep did express (Supplementary Figure 1). Though the fusion construct showed good expression, subsequent purification on a StrepTrap column produced a protein with very low purity. The eluent was pooled, diluted 10-fold in buffer (50 mM Tris HCl pH8, 300 mM NaCl, 10% glycerol and 1 mM TCEP) and injected to an equilibrated 5 ml HisTrap column. After washing three times with increasing imidazole concentration (20 - 80 mM), the protein was eluted using 250 mM imidazole with improved purity. The eluent was pooled and incubated overnight with TEV protease to cleave the His10-VLK portion, then passaged over a 1 ml His resin gravity column to elute CDK6-Strep in the flow through (Figure 6). The identity was confirmed using Western blotting with specific antibody and the obtained protein was soluble and reasonably pure, albeit with low yield (0.05 mg/L, 70% Table 1). Given the difficulty of expressing CDKs without their cyclin protein partner, this result gave an initial indication that adding VLK fusion protein to shift the kinase pI could improve expression.
Expression and purification of HER2 kinase domain
HER2 receptor tyrosine kinase is a well-known oncogene, whose cancer signaling is modulated by the catalytically inactive (pseudokinase) receptor HER3. (20,21) We designed several constructs encompassing the HER2 kinase domain with different sequence lengths and purification tags, with and without a point mutation V956R located in the its C-terminal lobe (Table 1, Supplementary Table 1). V956R blocks homodimerization of HER2, thus decreasing its activity and toxicity, consequently improving expression. (22) The kinase domain of HER2 (residues 705-1029) expressed in SF9 insect cells to the level of 0.15 mg/L culture, however, longer constructs including part of the juxtamembrane segment located N-terminally to the kinase domain (JMB) and the C-terminal tail (residues 691-1255) did not express in SF9. We therefore compared expression for short and long constructs in P. pastoris to determine whether yeast would allow expression of HER2 containing these protein-protein interaction domains.
Codon usage of the wild-type HER2 gene was optimized for P. pastoris without changing the amino acid composition (GenScript). HER2 kinase domain with the JMB and full-length C-terminal tail (residues 691-1255), with or without the V956R mutation and VLK-N-terminal sequence, was then expressed in P. pastoris. With a one-step purification on Strep-Trap columns, wildtype HER2 expressed at a modest but reproducible level and purity (0.2 mg/L culture, 70%), with some aggregation (Figure 7). Adding the VLK fusion protein eliminated the aggregation but was expressed at lower yield and purity (0.05 mg/L culture, 40%); V956R mutant HER2 expressed at apparent yields comparable to wild type but with lower purity (40%) as manifested by the higher molecular weight aggregates that could not be eliminated in subsequent chromatography steps. By size exclusion chromatography the wild type construct separated in agreement with it being a monomer, and its identity was confirmed using SDS-PAGE and Western blotting with anti-HER2 antibody. Both the wild type and mutant HER2 had kinase activity comparable to their commercial counterparts (Table 1).
Expression of the shorter HER2 construct with the kinase domain lacking the C-terminal tail (residues 705-1029) proved more challenging. While SF9 insect cells had produced high purity, active, protein in a monomer form with acceptable yield, this construct expression in P. pastoris suffered from aggregation, low yield and purity (Supplementary Table 1 and Supplementary Figure 2). Hence, SF9 and P. pastoris gave opposite results, with the long construct that encompasses almost entire intracellular portion of the receptor behaving better in yeast cells and kinase-domain only constructs expressing better in insect cells.