Figure 1.
Expression of GrB in the yeast Pichia pastoris.
(A) Constructs for expression of human granzyme B, and MBP-GrB and GST-GrB fusion proteins. AOX1, methanol-inducible alcohol oxidase I promoter; SP, α-factor signal peptide; furS, furin recognition motif; M, Myc tag; H, polyhistidine tag. (B) Culture supernatants of yeast clones carrying pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected after induction with methanol for 3 days, and expression of GrB was analyzed by immunoblotting with GrB-specific antibody. Each lane represents an individual clone. Clones marked with an asterisk were used in subsequent experiments. (C) For comparison, culture supernatants of clones carrying the different expression constructs were analyzed together in a single immunoblot experiment as indicated. Supernatant of a yeast clone carrying empty pPIC9 served as a control.
Figure 2.
Analysis of GrB secreted into the culture medium.
(A) Supernatants of yeast cultures harboring pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected at the indicated time points after induction with methanol, and analyzed by immunoblotting with GrB-specific antibody. Band intensities were quantified by determining mean gray values (MGV) relative to the highest value obtained. (B) Expression of an ErbB2 protein fragment. Yeast cells carrying pPIC9-ErbB2222 or pPIC9-MBP-furS-ErbB2222 were induced with methanol, and ErbB2 protein in culture supernatants was analyzed by immunoblotting with ErbB2-specific antibody.
Figure 3.
Growth kinetics of yeast clones and GrB mRNA expression.
(A) Cultures of P. pastoris carrying pPIC9-GrB, pPIC9-GST-furS-GrB, pPIC9-MBP-furS-GrB, or empty pPIC9 vector were induced with methanol. Samples were taken every 24 h, and OD600 was determined as a measure for cell density. (B) Quantification of GrB mRNA levels. RNA was isolated from yeast cells carrying pPIC9-GrB, pPIC9-GST-furS-GrB, pPIC9-MBP-furS-GrB, or empty pPIC9 vector after induction with methanol for one day. cDNA was synthesized and analyzed by quantitative real-time PCR including GAPDH for normalization. Means of duplicate samples are shown. Error bars indicate SD.
Figure 4.
Secretion of GST and MBP into the culture medium.
The presence of GST and MBP in yeast culture supernatants after induction for the indicated time periods was analyzed by immunoblotting with GST-specific (A) or MBP-specific antibodies (B). (C) Intracellular accumulation of GrB and GrB fusion proteins was investigated by analysis of cell lysates with GrB-specific antibody. The positions of the different GrB proteins are indicated by arrows. Samples analyzed were from the same cultures examined for secretion of GrB as shown in Fig. 2A.
Figure 5.
Processing of MBP-GrB fusion proteins.
(A) Variants of the furin recognition motif either containing (furS) or lacking (fur) an additional C-terminal serine residue. (B) Culture supernatants of yeast clones carrying pPIC9-MBP-fur-GrB were collected after induction with methanol for 3 days, and expression and processing of the GrB fusion protein was analyzed by immunoblotting with GrB-specific antibody. (C) Samples enriched for processed (100 kDa filtrate) or unprocessed MBP-fur-GrB protein (100 kDa retentate) were prepared by filtration of culture supernatant of a representative MBP-fur-GrB clone through Amicon filters with 100 kDa membranes, and analyzed by immunoblotting with GrB-specific antibody. Initial MBP-fur-GrB containing supernatant and supernatant from an MBP-furS-GrB clone were included for comparison. Supernatant of yeast cells carrying empty pPIC9 served as a control. The lower apparent molecular weight of processed GrB in (C) when compared to (B) is most likely due to a lower degree of glycosylation (calculated molecular weight for processed GrB in non-glycosylated form: 28.4 kDa). (D) Enzymatic activity of GrB proteins from culture supernatants and kinetics of substrate cleavage were analyzed in a colorimetric peptide cleavage assay. The MBP-fur-GrB or MBP-furS-GrB containing supernatants, and the MBP-fur-GrB samples enriched for processed (100 kDa filtrate) or unprocessed protein (100 kDa retentate) analyzed in (C) were tested as indicated. A purified recombinant GrB derivative (GrB) was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 displayed no GrB activity (data not shown). Means of triplicate samples are shown. Error bars indicate SEM.
Figure 6.
Processing and enzymatic activity of MBP-GST fusion proteins.
(A) Culture supernatants of representative yeast clones carrying pPIC9-MBP-fur-GST or pPIC9-MBP-furS-GST were collected after induction with methanol for 3 days, and expression and processing of the MBP-GST fusion proteins was analyzed by immunoblotting with GST-specific (left panel) or MBP-specific antibody (right panel). Supernatant of yeast cells carrying empty pPIC9 served as a control. (B) Enzymatic activity of MBP-GST fusion proteins and kinetics of conjugation of glutathione to synthetic CDNB substrate was analyzed in a colorimetric assay. The MBP-fur-GST or MBP-furS-GST containing supernatants analyzed in (A) were tested as indicated. Commercially available recombinant GST was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 served as negative control. Means of triplicate samples are shown. Error bars indicate SEM.