Cloning and expression of SARS-CoV-2 RBD in the yeast Pichia pastoris

Research Protocol: Cloning and Generation of Research Seed Stocks for SARS-CoV-2 RBD219 (Wild-type) Expressed in Pichia pastoris X-33

1.0       PURPOSE

This reportsummarizes the cloningof DNA encoding SARS-CoV-2-RBD219 WT and SARS-CoV-2-RBD219-N1 (deletion of the first Asn responsible for N-glycosylation) into yeast expression vector pPICZαA with and without a His-tag expressed at the C-terminus as well as transformation into Pichia pastoris X-33, small-scale expression screening to identify the expression yield, and selecting the clone with the highest expression yield from each construct to generate the research seed stocks.

2.0     SUMMARY

SARS-CoV-2 RBD219 is a 219 amino-acid receptor binding domain of SARS-CoV-2, the virus causing coronavirus disease-2019 (COVID-19), a potential candidate for vaccine development against COVID-19.

In order to express recombinant SARS-CoV-2 RBD219 protein for vaccine development, the yeast (P. pastoris) system was chosen as the expression platform. The DNA coding for SARS-CoV-2- RBD219-WT was codon optimized based on yeast codon usage preference and synthesized, then cloned into yeast expression vector pPICZαA with and without His-tag expressed at C-terminus. The recombinant plasmid DNAs was transformed into Pichia pastoris X-33. After being induced with 0.5% methanol in 10 mL medium, one clone with highest expression from each construct was selected for making research seed stocks.

3.0       MATERIALS

Pipettes (Pipetman® P1000, P200, P100, P20, P10 or equivalent)

0.1-10 μl Natural Tip (USA Scientific®, Cat# 1111-3800, or equivalent) 

1-200 μl Yellow tip (USA Scientific®, Cat# 1111-0806, or equivalent) 

1000 μl blue tip (USA Scientific®, Cat# 1111-2821, or equivalent)

SealRite 1.5ml Natural Microcentrifuge Tubes (USA Scientific®, Cat# 1615-5500, or equivalent) SealRite 0.5ml Natural Microcentrifuge Tubes (USA Scientific®, Cat# 1615-0000, or equivalent)

1.2 ml sterile cryovial (Nalgene, Cat# 5000-0012 or equivalent)

Serological Pipettes (25 ml Fisher Sci., Cat# 13-668-2, 10 ml Fisher Sci., Cat# 13-668-6, 1 ml Fisher Sci.,

Cat# 13-675-12B or equivalent).

50ml conical tubes (Falcon, Cat# 35-2098 or equivalent) 

15ml conical tubes (Falcon, Cat# 35-2095 or equivalent) 

Petri dishes (Fisher Scientific, Cat# 08-757-12 or equivalent) 

Pipette Aid (Drummund or equivalent)

500ml Erlenmeyer Flask with 0.2um vent cap (BioExpress, Cat# F-5909-500 or equivalent) 

1000ml Erlenmeyer Flask with 0.2um vent cap (BioExpress, Cat# F-5909-1000 or equivalent) 

VWR® Polypropylene Cell Spreaders, Sterile (VWR, Cat#89042-018 or equivalent)

0.2 µm Corning® Bottle-Top Filters, Sterile, 1000 ml (Corning, Cat#431174)

0.2 µm Corning® Bottle-Top Filters, Sterile, 500 ml (Corning, Cat#431118)

Corning® 1000 mL Vacuum Filter/Storage Bottle System, 0.22 µm (Corning, Cat# 430186) 

Corning® 500 mL Vacuum Filter/Storage Bottle System, 0.22 µm (Corning, Cat# 430758) 

Corning® 250 mL Vacuum Filter/Storage Bottle System, 0.22 µm (Corning, Cat#430756) 

CELLSTAR, CELLreactor™ 50 mL Tube (Greiner bio-one, Cat#227245 or equivalent)

2.5L TUNAIR™ CultureFlasks with full baffle (IBI Scientific, Cat#SS8003 or equivalent) Deionized water (Millipore® Elix system Texas Children’s Hospital)

Vector pPICZαA (Invitrogen, Cat# V195-20)

Restriction enzyme: Sac I (NEB,Cat # R0156M or equivalent) or BstX I (NEB, Cat# R0113L or equivalent) and Cutsmart Buffer (NEB, Cat # B7204S).

Pichia pastoris Strain X33 (Invitrogen, Cat# 50-0090/CI8000) Zeocin (Invitrogen, Cat# 45-0430 or equivalent)

Yeast Extract (Becton Dickinson and company, Cat# 212750 or equivalent) Bacto Peptone (Becton Dickinson and company, Cat# 211677 or equivalent) Yeast Nitrogen Base (Invitrogen, Cat# Q 300-09 or equivalent)

D-glucose Anhydrous (MP Biomedicals, Cat# 152527 or equivalent)

D-Sorbitol (Sigma, Cat# S-3889 or equivalent)

Bacto Agar (Becton Dickinson and company, Cat# 214050 or equivalent) YPD Agar (Becton Dickinson and company, Cat# 242720, or equivalent) Agarose (Invitrogen, Cat: 15510-027 or equivalent)

Orthophosphoric acid ≥85%, Finyte®, J.T. Baker® (Cat#JT5854-5, or equivalent)

Potassium hydroxide, Powder for synthesis MilliporeSigma (CAS Number: 1310-58-3, or equivalent)

50x TAE (Gibco, Cat: 24710-030 or equivalent)

2x SDS loading buffer (Invitrogen, Cat: LC2676 or equivalent) SeeBluePlus2 Prestained protein standard (Invitrogen, Cat:LC5925)

Novex® 4-20% Tris-Glycine gel 1.0 mm, 12 well (Invitrogen, Cat# EC60252BX5)

Nove® 4 to 20%, Tris-Glycine, 1.0 mm, Mini Protein Gel, 15-well (Invitrogen, Cat# XP04205BOX)

1 Kb Plus DNA ladder (Invitrogen, Cat: 10787-026)

BlueJuice Gel Loading buffer (Life Technologies™, Cat# 10816-015, or equivalent) Potassium phosphate monobasic (MW 136.09, Spectrum, Cat: P0200 or equivalent) Potassium phosphate dibasic (MW 174.18, Spectrum, Cat: P0240 or equivalent) 100% Methanol (Spectrum, Cat: ME151 or equivalent)

Ammonium hydroxide, 8N (J.T. Baker, Cat: 9726-02 or equivalent)

Biotin (MW 244, Sigma, Cat: B-4501 or equivalent) Glycerol (Macron, Cat# 5092-16 or equivalent)

Select Phytone Peptone (Soybean derived without animal source, BD, Cat# 210931) Gene Pulser cuvettes (2MM Cuvette W/IN, Cat# 1652086. BIO-RAD, Laboratories, CA)

UltraPure™  Phenol:Chloroform:Isoamyl  Alcohol  (25:24:1,  v/v)  (Invitrogen, Cat# 15593- 031 or equivalent)

200 Proof Ethanol (Fisher Scientific, Cat#NC9189167 or equivalent) Sodium Acetate Buffer Solution (Sigma, Cat# S7899 or equivalent) Sodium Hydroxide, 10N Solution (J.T.Baker®, Cat#5000-02 or equivalent)

SARS-CoV-2 Spike Ig Rabbit Mab (1mg/mL), SinoBio, Lot: 40150-R007 THETM His Tag

Antibody, mAb, Genescript , Lot: 18F001522

4.0   STUDY DESIGN/METHODS

4.1 RBD background

The SARS-CoV-2-RBD219 is a 219 amino acid receptor binding domain derived from the spike protein (GenBank: QHD43416.1) of severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 (GenBank: MN908947.3). It contains 219 amino acids with a theoretical pI/Mw of

8.67 / 25591.76 (including a 6His-tag).

4.2 DNA synthesis

The coding DNA sequence for SARS-CoV-2-RBD219-WT was codon optimizedbased on yeast usage preference, and then synthesized by GenScript

Yeast codon optimized DNA sequence for SARS-CoV-2-RBD219-WT: 
AACATCACTAATTTGTGTCCATTCGGTGAAGTTTTTAATGCTACAAGATTCGCATCAGTT TACGCTTGGAACAGAAAGAGAATCTCAAACTGTGTTGCAGATTACTCTGTTTTGTACAAC TCTGCTTCATTTTCTACTTTTAAATGTTACGGTGTTTCACCAACTAAGTTGAACGATTTG TGTTTCACAAATGTTTATGCAGATTCTTTTGTTATTAGAGGTGACGAAGTTAGACAAATT GCACCAGGTCAAACTGGTAAAATCGCTGATTACAACTACAAGTTGCCAGATGATTTCACA GGTTGTGTTATCGCTTGGAACTCTAACAATTTGGATTCTAAGGTTGGTGGTAACTACAAC TACTTGTACAGATTGTTTAGAAAATCAAATTTGAAGCCATTCGAAAGAGATATCTCAACT

GAAATCTATCAAGCAGGTTCTACACCATGTAACGGTGTTGAAGGTTTTAATTGTTACTTC CCATTGCAATCTTATGGTTTTCAACCAACAAATGGTGTTGGTTACCAACCATACAGAGTT GTTGTTTTGTCATTCGAATTGTTACATGCTCCAGCAACTGTTTGTGGTCCAAAGAAATCT 
ACAAATTTGGTTAAAAATAAGTGTGTTAACTTCAACTTCAATGGTTTAACTGGTACA

.

4.3 Expression vector

pPICZαA (Invitrogen, Cat# V195-20) is a 3.6 kb vector used to express and secrete recombinant proteins in P. pastoris (Figure 3A). Recombinant proteins are expressed as fusions to an N-terminal peptide encoding the Saccharomyces cerevisiae α-factor secretion signal “MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA” (Figure 1B). The vector allows high-level, methanol inducible expression of the gene of interest in Pichia, and can be used in any Pichia strain including X-33.

Figure 1 (A) The map of pPICZαA vector (www.snapgene.com). (B) The cloning sites and the DNA coding

signal sequence in pPICZαA.

4.4 Cloning of DNAs coding for SARS-CoV-2-RBD219-WT and SARS-CoV-2-RBD219N1 into the expression vector

The coding DNAs for SARS-CoV-2-RBD219-WT, with and without 6His-tag at C-terminus, were cloned into yeast expression vector pPICZaA using EcoRI and XbaI sites, by GenScript.

4.5 Expression yeast strains

Pichia pastoris X-33 (Invitrogen, Cat# K1740-01) was used to express recombinant SARS- CoV-2 RBD219 and SARS-CoV-2 RBD219-N1 proteins. P. pastoris X-33 is a methylotrophic yeast, capable of metabolizing methanol as its sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde using molecular oxygen by the enzyme alcohol oxidase. In addition to formaldehyde, this reaction generates hydrogen peroxide. To avoid hydrogen peroxide toxicity, methanol metabolism takes place within a specialized cell organelle, called the peroxisome, which sequesters toxic by-products away from the rest of the cell. Alcohol oxidase has a poor affinity for O2, and Pichia pastoris compensates by generating large amounts of the enzyme. The promoter regulating the production of alcohol oxidase is the one used to drive heterologous protein expression in Pichia. The detailed information of pPICZ™ expression vector and the yeast strain is available from the manufacturer: EasySelect™ Pichia Expression Kit, Rev. Date 18 June 2010, Manual part no. 25-0172 (Invitrogen).

4.6 Transformation of recombinant plasmid DNA into yeast competent cells

Two recombinant plasmids: SARS-CoV-2-RBD219-WT+His/pPICZaA and SARS-CoV- 2RBD219-WT/pPICZaA were transformed into P. pastoris X-33 as described in the Invitrogen EasySelect™ Pichia Expression Kit Manual. Briefly, the recombinant plasmid DNAs were linearized with SacI. The linearized plasmid DNAs were transformed into P. pastoris X-33 using electroporation. The transformants were selected on YPD plates with 100 µg/mL and 500 µg/mL of Zeocin for being incubated at 30°C for three days.
 

4.7 Induction of recombinant RBD and identification of the clone with highestexpression

After being incubated at 30°C for three days, 14 colonies from each construct were picked from transformation plates to inoculate 10 mL BMMY medium with 0.5% methanol at 30°C for three days for recombinant protein induction. The expressed recombinant proteins were identified with SDS-PAGE and Western blot with anti-His antibody (only for clones with His-tag). The clone with highest expression was selected from each construct transformation for making research seed stocks.
 

4.8 Generation of research seed stocks

The clones with highest expression from each construct transformation identified in the small-scale (10 mL) expression were selected for making the research seed stocks. Each selected clone was picked from the corresponding transformation plate to streak on a new YPD/Zeocin plate to grow at 30°C for 48-72 hours to make single colonies. One single colony from each plate was used to inoculate 20 mL of YPD medium. After overnight growth at 30oC, glycerol was added to the culture at a final concentration of 20%. After being well-mixed, the glycerol-culture mixture was aliquoted into cryovials at 1 mL and stored at -80°C as the research seed stocks.

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