The effect of the bgs13 mutation on the structure of the reporter protein beta-lactoglobulin: Influence on folding and aggregation in Pichia pastoris

Abstract

Pichia pastoris, a methylotrophic yeast used for recombinant protein expression, has the capability of performing many eukaryotic post-translational modifications, growing to high cell densities, and producing proteins in a cost-effective manner. However, P. pastoris's secretion properties are not always efficient, and its secretory pathway mechanisms have not been thoroughly elucidated. A previously identified mutant strain, bgs13, was found to efficiently secrete most recombinant proteins tested, raising the possibility that this bgs13 mutant is a universal super secreter. In this study, we used a reporter protein, β-lactoglobulin (b-LG), to perform structural analysis of the protein secreted from wild type and mutant bgs13 strains to investigate the secretory mechanism. Primary, secondary, and tertiary structures of b-LG were examined using Edman sequencing, circular dichroism, tryptophan fluorescence, and temperature induced aggregation analysis. Our results demonstrate that the bgs13 produced more b-LG than the wt strain and that this protein was functionally folded similar to the wt. Surprisingly, we also found that the bgs13 b-LG was more resistant to aggregation, providing another example of the superior qualities of this strain for enhanced secreted protein production.

Figure 1:

A. The secretory system of P. pastoris B. Plasmid map of pBI1.

Figure 2.

Coomassie staining of proteins in extracellular media of induced strains. Cultures were grown for 2 days in glucose medium, shifted to medium with methanol, and then harvested after 48 hours. Samples of extracellular media were concentrated about 5 fold, desalted and then resolved on SDS PAGE, and proteins were visualized with Coomassie blue stain. The indicated amounts of commercially available, native b-LG were run as a comparison. The diffuse 30 kD recombinant b-LG bands are indicated by the arrow.

Figure 3.

Western analysis of proteins in extracellular media of induced strains with an anti-c-myc antibody. Cultures were grown for 2 days in glucose medium, shifted to medium with methanol, and then harvested after 48 hours. Proteins of extracellular media were resolved on SDS PAGE, transferred to nitrocellulose, and probed with an anti-c-myc antibody. The negative control was a strain containing the empty pBLHIS SX plasmid while S123 strain produces the hyperglycosylated b-LG without the c-myc and his6 tags. Two representative cultures were chosen to demonstrate the expression of b-LG by both the wt and bgs13 strains.

Figure 4.

Western analysis of proteins in extracellular media of induced strains with an anti-b-LG antibody. Cultures were grown for 2 days in glucose medium, shifted to medium with methanol, and then harvested after 48 hours. Proteins of extracellular media were resolved on SDS PAGE, transferred to nitrocellulose, and probed with an anti-b-LG antibody. The negative control was a strain containing the empty pBLHIS SX plasmid while S123 strain produces the hyperglycosylated b-LG without the c-myc tag. Two representative cultures were chosen to demonstrate the expression of b-LG by both the wt and bgs13 strains.

Figure 5.

SDS PAGE of samples from b-LG purification. Equal volumes from the various steps in the purification of the b-LG from the extracellular media were subjected to SDS-PAGE and then visualized with Coomassie stain. Lanes 1–4 are from the bgs13:pBI1 culture while lanes 5–8 are from the wt:pBI1 culture. Lane 9 contains1 ug commercial b-LG; lane 10 contains molecular weight ladder.

Figure 6.

EndoHf assay of the wt and bgs13 b-LG proteins with Rnase B. bgs13+= bgs13 b-LG treated with EndoH; bgs13−= bgs13 b-LG with no EndoH, wt+= wt b-LG treated with EndoH; wt−= wt b-LG with no-EndoH; RnaseB+ =Rnase B control treated with EndoH; RnaseB−= Rnase B control with no EndoH; EndoH= EndoH alone. The bands containing EndoH digested b-LG and the EndoH are indicated in the bgs13+ lane.

Figure 7:

Predicted amino acid sequence at the N-terminus of b-LG. The bold arrow indicates where the MATα signal peptide should have been cleaved by a Kex2 protease. The Ste13 protease digests on the C terminal side of Glu-Ala (indicated by a star); however, its activity is less reliable.

Figure 8:

CD spectrum of b-LG samples. CD spectra (200–260nm) showing commercial b-LG along with the b-LG produced from wt and bgs13, respectively.

Figure 9:

CD absorbance at 215nm of b-LG from wt and bgs13 as a function of temperature increase.

Figure 10.

CD absorbance at 207nm of b-LG from wt and bgs13 as a function of temperature increase. b-LG transition curves were fit using the Sigmoidal 4-PL fit in GraphPad Prism to determine the transition temperature (Tm). Both wt and bgs13 curves start at −6 mdeg which confirms that they were at the same concentration.

Figure 11.

Trp fluorescence as a function of wt and bgs13 b-LG. Trp fluorescence was measured at 340 nm; background changes due to thermal dependence of Trp fluorescence intensity were subtracted by using a linear slope determined from the range outside the transition, and the data was normalized for fraction unfolded. To determine the transition temperature (T_m), we used the sigmoidal fit in GraphPad PRISM.

Figure 12:

SDS PAGE of SDS-PAGE gel examining aggregation of b-LG at different temperatures. Wt and bgs13 samples of b-LG were heated for 5 minutes at the indicated temperatures to induce aggregation followed by the immediate addition of stop solution containing iodoacetamide to block free sulfhydryls. Samples were run under nonreducing conditions. Monomer (non-aggregated) and dimer (aggregated) forms are indicated by arrows.

Figure 13.

SDS PAGE of wt and bgs13 b-LGs heated at 65°C for various times. Wt and bgs13 samples of b-LG were heated for 3 and 18 hours at 65°C followed by additions of stop solution containing iodoacetamide to block free sulfhydryls. Samples were run under nonreducing conditions. Monomer (non-aggregated) and dimer and large polymer (aggregated) forms are indicated by arrows.

Figure 14.

Measurement of free sulfhydryls in wt and bgs13 b-LG. A standard curve was created based on the assays which measure the fluorescence produced by known concentrations of commercial b-LG. 2 μM and 4 μM concentrations (based on OD₂₈₀) of wt and bgs13 b-LG were plotted against the curve.