Sep 04, 2020

Public workspaceRecombinant protein expression and purification of codon-optimized Bst-LF polymerase

DOI
dx.doi.org/10.17504/protocols.io.bksrkwd6
  • 1Institute for Biological and Medical Engineering, Pontificia Universidad Católica de Chile;
  • 2Millennium Institute for Integrative Biology (iBio), Santiago, Chile;
  • 3National Jewish Health;
  • 4FCEN-Universidad de Buenos Aires, Argentina
  • Maira Rivera: Millennium Institute for Integrative Biology (iBio), Santiago, Chile;
  • Reclone.org (The Reagent Collaboration Network)
  • XPRIZE Rapid Covid Testing
Cesar A Ramirez-Sarmiento
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DOI: dx.doi.org/10.17504/protocols.io.bksrkwd6
Protocol Citation: Maira Rivera, Severine Cazaux, Ariel Cerda, Anibal Arce Medina, Isaac Núñez, Tamara Matute, Alex Brown, Javier Gasulla, Fernan Federici, Cesar A Ramirez-Sarmiento 2020. Recombinant protein expression and purification of codon-optimized Bst-LF polymerase. protocols.io https://dx.doi.org/10.17504/protocols.io.bksrkwd6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our workspace and it is working. Typical protein yield is about 20 mg per liter of cell culture. Functionally tested in a LAMP assay using both commercial and in-house buffers equivalent to Bst2.0 from New England Biolabs and an initial protein concentration of 0.25 ug/ml (8-fold dilution from the initial 2 mg/ml stock). LAMP and RT-LAMP Assay protocols soon to be published.
Created: September 03, 2020
Last Modified: September 04, 2020
Protocol Integer ID: 41521
Keywords: RT-LAMP, isothermal amplification, COVID-19, SARS-CoV-2,
Abstract
This protocol has been optimized for the recombinant expression of a codon-optimized Bst-LF polymerase.

The goal of this protocol was to eliminate the use of large volumes for dyalisis through the use of concentrators for buffer exchange before storage conditions.

The plasmid encoding the codon-optimized Bst-LF enzyme used here can be found on reclone.org
Materials
MATERIALS
ReagentAmicon Ultra-15 Centrifugal Filter UnitEmd MilliporeCatalog #UFC910024
ReagentSodium phosphate monobasic monohydrateSigma AldrichCatalog #S9638
ReagentPMSFSigma AldrichCatalog #P7626
ReagentSodium phosphate dibasicSigma AldrichCatalog #7558-79-4
ReagentImidazoleSigmaCatalog #I5513
ReagentNaClSigma AldrichCatalog #53014
ReagentHisTrap FF Crude ColumnGe HealthcareCatalog #17528601
ReagentLysozymeThermo Fisher ScientificCatalog #89833
ReagentGlycerolMerck MilliporeCatalog #104092
ReagentDTTMillipore SigmaCatalog #DTT-RO
ReagentTriton X-100Sigma AldrichCatalog #X100-100ML
ReagentTrizma® baseMerck Millipore SigmaCatalog #93362
ReagentEDTASigma AldrichCatalog #ED2SS
ReagentKClSigmaCatalog #P9541
Buffer A, pH 8.0
Concentration50 millimolar (mM) NaPO4, pH 8.0
Concentration300 millimolar (mM) NaCl
Concentration30 millimolar (mM) Imidazole, pH 8.0

Buffer B, pH 8.0
Concentration25 millimolar (mM) Tris-HCl, pH 8.0
Concentration200 millimolar (mM) KCl
Concentration30 millimolar (mM) Imidazole, pH 8.0

Buffer C, pH 8.0
Concentration25 millimolar (mM) Tris-HCl, pH 8.0
Concentration100 millimolar (mM) KCl
Concentration150 millimolar (mM) Imidazole, pH 8.0

Buffer D, pH 8.0
Concentration25 millimolar (mM) Tris-HCl, pH 8.0
Concentration100 millimolar (mM) KCl

Storage conditions
Concentration12.5 millimolar (mM) Tris-HCl, pH 8.0
Concentration50 millimolar (mM) KCl
Concentration0.1 millimolar (mM) EDTA
Concentration0.1 % volume Triton X-100
Concentration50 % volume Glycerol
Concentration1 millimolar (mM) DTT

DAY 1 – Plasmid transformation
DAY 1 – Plasmid transformation
1d
1d
Transform Amount100 ng of pET15b plasmid containing codon-optimized Bst-LF polymerase into E. coli C41 competent cells using either heat shock or electroporation.
2h
Spread transformed cells in LB Agar plates supplemented with Concentration0.1 mg/mL Amp. Grow plate overnight at Temperature37 °C .
12h
DAY 2 – Preinoculum
DAY 2 – Preinoculum
1d
1d
Select a single colony from the LB agar plate to prepare a preinoculum in Amount10 mL LB media supplemented with Concentration0.1 mg/mL Amp. Grow overnight at Shaker250 rpm, 37°C .
1d
DAY 3 – Protein Overexpression
DAY 3 – Protein Overexpression
1d
1d
Use the full volume of the preinoculum to inoculate Amount1 L of LB media supplemented with Concentration0.1 mg/mL Amp (1% inoculation). Grow at Shaker200 rpm, 37°C until reaching an optical density at 600 nm (OD600) = 0.8.
4h
Upon reaching OD600 = 0.8, add IPTG to a final concentration of Concentration0.5 millimolar (mM) and incubate overnight at Shaker200 rpm, 18°C .
16h
DAY 4 – Protein Purification and Storage
DAY 4 – Protein Purification and Storage
6h
6h
Centrifuge the cell culture Centrifigation4000 x g, 4°C, 00:30:00 .Then, resuspend the cell pellet in Amount40 mL of Buffer A freshly supplemented with Concentration1.0 millimolar (mM) PMSF and Concentration0.2 mg/mL lysozyme.

30m
Incubate the resuspended cells Shaker80 rpm, Room temperature , 00:30:00 .
30m
Sonicate on ice for Duration00:08:00 using cycles of Duration00:00:01 ON and Duration00:00:04 OFF at 40% amplitude (Qsonica Q125, 125W).
30m
On an ultracentrifugation tube, incubate the unclarified lysate at Temperature65 °C for Duration00:25:00 to precipitate most of E. coli proteins, and then place on ice for Duration00:05:00 . Centrifuge Centrifigation20000 x g, 4°C, 00:30:00 and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.
1h
On a 5 mL HisTrap column (GE Healthcare) pre-equilibrated with 10 column volumes (c.v.) (here, 50 mL) of Buffer A, load the supernant. Wash with 10-20 c.v. of Buffer B. Then, elute with 5 c.v. of Buffer C, collecting the eluted fractions every Amount1 mL in 1.5 ml tubes.
1h
To quickly pool the fractions containing the protein of interest, prepare a 96-well plate or 1.5 mL tubes with Amount40 µL of 5X Bradford reagent and Amount160 µL of distilled water. Then, add Amount10 µL of each protein fraction and compare against a blank reference sample corresponding to Amount10 µL of Buffer B. You can determine your protein-containing fractions either by absorbance at 595 nm on a plate reader or visually by comparing the blue coloration of each fraction against the blank reference. Pool your fractions and collect a Amount10 µL sample for SDS-PAGE.
5m
To decrease the imidazole concentration, perform a buffer exchange step with an Amicon Ultra-15 concentrator (Merck Millipore). Centrifuge Centrifigation3000 x g, 10°C, 00:10:00 , discard the flowthrough, add Buffer D to decrease the imidazole concentration and repeat this step, until the imidazole concentration reaches < 30 mM.
1h
Recover the concentrated protein and determine its concentration using the Bradford assay. Then, supplement with Concentration0.2 millimolar (mM) EDTA, Concentration0.2 % volume Triton X-100, Concentration2 millimolar (mM) DTT and add glycerol up to Concentration50 % volume to reach Storage Conditions. Do consider that a final protein concentration ≤Concentration2 mg/mL is appropriate for subsequent experiments.
5m
Generate Amount200 µL aliquots of the enzyme and store it at Temperature-20 °C until required.
30m
IMAC SDS-PAGE Result
IMAC SDS-PAGE Result
SDS-PAGE at 10% PA of eluted fractions from IMAC purification of Bst-LF. The high MW contaminant is due to disulfide bond formation and eliminated upon addition of DTT (data not shown).