Difference between revisions of "Part:BBa K4365021"

Revision as of 09:18, 11 October 2022

SP-SUMO-turboRFP

SP-SUMO-tRFP is a reporter device which could be used to test protein secretion in yeast. Moreover, if tRFP is exchanged for the protein of interest, after protein purification with His-tag you can scarlessly remove the tag from the protein with a SUMO protease. This part is optimized for expression in Saccharomyces cerevisiae.

SP-SUMO-tRFP consists of:

• FIG1 promoter K4365000
• signal peptide (alpha mating factor signal peptide) K4365019
• glycine linker K4365005
• 6x His-Tag K3033006
• SUMO K4365004
• codon optimized turboRFP K4365020
• terminator tCYC1 K849009

Figure 1: illustration of SP-SUMO system for secretion and scar-less protein production in yeast. The SP-SUMO is attached to the cargo protein turboRFP as this was the construct we tested during our experiments. Created with Biorender.com.

Sequence and Features

Usage and Biology

Figure 2: Alpha fold structural prediction of the SP-SUMO construct. During the design phase, Alpha fold helped us visualize the structure. SUMO (Pink) Glycine linker (green), His-tag (red), Alpha Secretion factor (blue).

SP-SUMO system was designed by TU_Dresden22. It is a system for “scar-less” protein production in yeast which consists of a N-terminal SP, a His tag, and a SUMO tag. A Glycin-Serin flexible linker was inserted between the alpha-mating factor prepro peptide signal peptide and the His tag for two reasons: to shield the signal peptide from possible effects due to the changes of the His tag and to give the SRP more time to find and block the translating ribosome to increase the chances of the nascent peptide to safely reach the ER before the SUMO is cleaved in the cytoplasm.

This system should not leave any residual amino acids after cleavage and would provide proteins in their optimal native structure. SP-SUMO is a modular protein system. The His tag can be exchanged for other tags and the signal peptide can be exchanged with more the high-efficiency signal peptides.

Cloning strategy

Proof of SP-SUMO secretion

After transformation into yeast, the SP-SUMO turboRFP construct and the turboRFP construct were tested following the secretion assay protocol in flasks. The turboRFP construct was employed as a control for cell lysis. The two cultures were induced with 500 nM Alpha-Factor Mating Pheromone (Zymo research). Two additional cultures were adjusted to 1 OD and were not induced and grown as controls.

The growth of the four cultures was monitored over the course of several hours by measuring their optical density and taking samples of supernatant. The supernatant fluorescence intensity was measured with a plate reader and graphs of OD and Fluorescence normalized over OD were plotted (Figure 5).

Figure 5: secretion assay in flasks of the SP-SUMO-turboRFP from 1 to 28 hours after induction. (A) Growth curves of the transformed yeast. (B) Fluorescence normalized over OD. The construct was cloned into the p426 shuttle plasmid. The control used to account for cell lysis was the turboRFP without SP-SUMO. The SP-SUMO-turboRFP and the turboRFP control were grown in an uninduced state and an induced state (+alpha)

For the first experiment, the fluorescence measurements were taken starting at 1 hr post-induction (hpi) until 28 hpi. We decided to take more measurements from 12 hpi to 22 hpi as this was shown to be the interval of time in which most of the secretion had taken place in the first data acquired.

Figure 6: secretion assay in flasks of the SP-SUMO-turboRFP from 12 to 22.5 hours after induction. (A) Growth curves of the transformed yeast. (B) Fluorescence normalized over OD. The construct was cloned into the p426 shuttle plasmid. The control used to account for cell lysis was the turboRFP without SP-SUMO. The SP-SUMO-turboRFP and the turboRFP control were grown in an uninduced state and an induced state (+alpha).

The experiments showed that, after induction of the FIG1 promoter, the cells containing the SP-SUMO turboRFP construct secreted more turboRFP into their medium than the construct expressing turboRFP and the two uninduced controls. Moreover, even if a certain level of cell lysis was present due to cell death and turnover, the difference in turboRFP secretion was still clear.

After assembling the SP-SUMO-turboRFP construct in the pSB1KY plasmid we performed a 48-well plate secretion assay.

This experiment was performed twice to verify that the data was solid. The results confirmed that the SP-SUMO construct was able to secrete turboRFP in the medium. In addition to the negative control for cell lysis, a construct consisting of the alpha mating factor signal peptide aminoterminally fused to the turboRFP protein was used as a positive control. Strikingly, the secretion of the SP-SUMO is considerably higher than this positive control. This finding confirms what is reported in the literature regarding the effects of SUMO on protein production ^[1].

Figure 7: secretion assay in 48-plates of the SP-SUMO-turboRFP. The supernatant fluorescence is normalized over nephelometric turbidity units. The construct was expressed from the p426 (blue) and pSB1KY shuttle plasmid (orange). The control used to account for cell lysis was the turboRFP without SP-SUMO (grey and brown). A construct consisting of the alpha mating factor signal peptide fused to the turboRFP protein was used as a positive control (black). The SP-SUMO-turboRFP and the turboRFP control were grown in an induced state (+alpha) only.

Secretion assay in a flask protocol

Figure 3: graphical presentation of the workflow of the signal peptide secretion assay in 48-well plate. Created with Biorender.com.

After transformation into yeast, the signal peptide-containing constructs were grown overnight in MV medium at 30°C in a shaking incubator at 180 RPM. The following day, a 20 mL culture was prepared by adjusting the overnight cultures to 1 OD so that sufficient biomass was available for protein production. The culture was then induced with 500 nM alpha mating factor pheromone (Zymo research) and grown at 30°C in a shaking incubator at 180 RPM.

The growth of the cultures was monitored over the course of several hours by measuring their optical density using a spectrophotometer. Due to the size of the yeast cells, it was always necessary to perform a 1:10 dilution of the yeast culture before measuring the OD. The supernatant samples containing the secreted protein of interest were taken after centrifugation of 1 mL of yeast culture at 3.500 x g and snap-frozen with liquid nitrogen.

The supernatant samples were rapidly thawed and pipetted onto a 96 well-plate (Corning 96-well plates, flat bottom, black) and measured in the Tecan pro infinite 2000 (gain: 120, excitation wavelength: 553 nm, wavelength: 620 nm).

Secretion assay in a 48-well plate protocol

Figure 4: graphical presentation of the workflow of the signal peptide secretion assay in 48-well plate. Created with Biorender.com.

After transformation into yeast, the signal peptide-containing constructs were grown overnight in MV medium at 30°C in a shaking incubator at 180 RPM. The following day, a culture is prepared by adjusting the overnight cultures to 1 OD in MV medium.

After induction of the culture with 500 nM alpha mating factor pheromone, 1 mL was pipetted into 3 wells of a transparent 48-well plate (Greiner Bio-one CELLSTAR, 48-well Plate) to create biological triplicates. A turboRFP alpha mating factor signal peptide fusion protein was added as positive secretion control, the turboRFP without signal peptide as a negative secretion control. A blank was also added to all of the plates.

A permeable membrane (Sigma-Aldrich, Breathe-Easy sealing membrane) was used to seal the plates and allow gas exchange. As many plates were set up as the number of planned time points to be measured because the permeable membrane needs to be removed to conduct the measurement and the yeast culture is contaminated. The plates were then placed at 30°C in a shaking incubator at 220 RPM.

Every time the secretion had to be measured, a plate was taken out of the incubator, the permeable membrane was removed and the growth of the cultures measured by turbidity using a nephelometer (NEPHELOstar, BMG LABTECH). The plate was then centrifuged at 35000 xg for 10 minutes and 200 µL the supernatant samples containing the secreted protein were taken avoiding the carryover of any yeast cells. The supernatant sample was pipetted into a 96 well-plate (Corning 96-well plates, flat bottom, black) and measured in the Tecan pro infinite 2000 (gain: 120, excitation wavelength: 553 nm, wavelength: 620 nm).

Proof of His-tag functionality

Figure 8: SDS-PAGE gel of the pellet of SP-SUMO-turboRFP expressing yeast cells, before and after batch binding (BB). The lysed pellet, the supernatant of the culture, the lysed pellet after batch binding, and the supernatant after batch binding were examined. Protein Ladder= Thermo Scientific PageRuler Plus Prestained Protein Ladder.

After verifying that the construct was secreted into the growth medium, we performed batch binding purification on a 500 ml yeast culture grown in MV medium for 3 days. The pellet obtained after centrifugation and the supernatant were used for [batch-binding purification](https://www.notion.so/1bac1a97cb4343c1ad46760e40e2489f).

Four samples were obtained from the purification procedure: lysed pellet, supernatant, lysed pellet after batch binding, and supernatant after batch binding. These samples were used to run [an SDS/Page gel and a Western blot](https://www.notion.so/Protein-Analysis-0d67ad0091fd4f28918ebe033534c611) using antibodies against turboRFP and the His6 tag.

The Coomassie stain (Figure 8) showed that the supernatant contained fewer secreted proteins compared to the pellet and indicated that indeed a secreted protein would already possess a high degree of purity relative to a protein extracted by cell lysis.

The Western blot revealed that the full SP-SUMO turboRFP construct was present in the transformed yeast cells (Figure 9). Two protein bands were visible on the lane corresponding to the lysed pellet of cells. One band at ~25 kDa corresponds to the turboRFP size. The band at ~60 kDa, we hypothesize, corresponds to the SP-SUMO-turboRFP (~25 kDa + 13 kDa) bound to the Ulp1 protease (~29 kDa). This indicated that a good portion of the protein was already cleaved before leaving the cell. Sill the supernatant contained secreted turboRFP without SP-SUMO (Figure 9). The batch binding purification was able to capture the SP-SUMO from the lysed pellet but not from the supernatant.

Figure 9: Western blot with anti-turboRFP (A) and anti-His6 antibodies (B). The lysed pellet, the supernatant of the culture, the lysed pellet after batch binding (BB), and the supernatant after batch binding (BB) were analyzed. Protein Ladder= Thermo Scientific PageRuler Plus Prestained Protein Ladder.

References

1. ↑ Butt TR, Edavettal SC, Hall JP, Mattern MR. (2005) SUMO fusion technology for difficult-to-express proteins, Protein Expr Purif. ;43(1):1-9. doi: 10.1016/j.pep.2005.03.016. Epub 2005 Apr 9. PMID: 16084395; PMCID: PMC7129290.