Fastdigest restriction endonucleases

Genomic and plasmid DNA isolations were performed with Bacterial & Yeast Genomic DNA Purification Kit (EURx Sp. z o.o., Gdańsk, Poland) and Plasmid Miniprep DNA Purification Kit (EURx Sp. z o.o., Gdańsk, Poland), respectively, according to the manufacturer’s protocols. Molecular cloning and transformation were performed according to standard protocols [70 ]. FastDigest restriction endonucleases were purchased in Thermo Fisher Scientific (Waltham, MA, USA). PCR was performed with high-fidelity Platinum SuperFi II DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations. The plasmids constructed and used in this work are listed in Supplementary Tables S2, S4, and S6–S8. The primers used in this work were synthesized at Genomed S. A. (Warsaw, Poland) and are listed in Supplementary Tables S1, S2, and S5–S7. Sanger DNA sequencing of plasmid constructs prepared in this work was performed in Genomed S. A. (Warsaw, Poland).

E. coli DH5α was used for propagation of plasmids and E. coli BL21 (DE3) was for expression of the α-galactosidase gene. The pET28a(+) (Merck, Darmstadt, Germany) vector was used for gene cloning. Fastdigest restriction endonucleases and T4 DNA ligase were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Plasmid extraction kits and Bacteria Genomic DNA kits were procured from Tiangen (Beijing, China). Ni²⁺-NTA agarose was purchased from Qiagen (Hilden, Germany) to collect His-tagged protein. Proteinase K and trypsin were from Amresco (Solon, OH, USA), and subtilisin A and α-chymotrypsin were provided by Sigma (St. Louis, CA, USA). The centrifugal filter used to concentrate protein and the silica gel plates used for thin-layer chromatography (TLC) analysis were purchased from Merck (Darmstadt, Germany). Unless otherwise stated, all other chemicals and reagents used in this paper were of analytical grade.

Genomic DNA was extracted from infected erythrocytes [40 ] and used for the amplification of various genes for both recombinant protein expression and transgenic parasite production. The primers, containing appropriate restriction endonuclease sites, are presented in the Additional files 1 and 2. PCR products and vectors were digested with the appropriate FastDigest^® restriction endonucleases (Thermo Fisher Scientific, Inc., Ueberlingen, Germany). The genes or gene domains were subsequently cloned into pARL2-GFP (donated by Dr Jude Przyborski, Marburg, Germany), pGEX-4T-2 or pET-15b vectors depending on the downstream application. After ligation, the pARL2-GFP constructs were used for the transformation of Escherichia coli XL10-Gold^® ultracompetent cells (Stratagene, CA, USA), while the Rosetta™ 2 (DE3) Escherichia coli line (Novagen Inc., WI, USA) was used for the pGEX-4T-2 and pET-15b constructs.

The pEU3-NII vector backbone was previously modified by Bardóczy et al. [5 (link)] to encode for His₆-tag (pEU3-NII-HLIC) and GST-tag (pEU3-NII-GLIC). The NotI restriction endonuclease was introduced into both vectors using QuikChange Site-Directed Mutagenesis Kit (Agilent) according to the provided manual with the 5′-ATTCTGAGAATAGTGTATGCGGCcgCCGAGTTGCTCTTGCCCGG-3′ and 5′-CCGGGCAAGAGCAACTCGGcgGCCGCATACACTATTCTCAGAAT-3′ primers (NotI recognition sequence underlined, mutated bases lowercase). The resulting pEU3-NII-HLICNot and pEU3-NII-GLICNot vectors were further manipulated to produce novel modified vectors suitable for wheat germ cell-free protein expression.
In general, the previously constructed pEU3-NII-HLICNot and pEU3-NII-GLICNot vectors were cut with the appropriate FastDigest restriction endonucleases (Thermo Scientific), treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and used in ligation reactions. The colonies containing the new fragments were selected by colony PCR with pEU3-NII sequencing primers pEU3-NII-forward (5′-CACTATAGGGTACACGGAATTCGC-3′) and pEU-rev (5′-TATAGGAAGGCCGGATAAGACG-3′). Colony PCR positive clones were propagated, and the plasmid DNAs were isolated with Zippy Plasmid Miniprep kit (Zymo Research) for sequencing. See Supplementary Figure 1, Additional file 1 for the schematic representations of vector constructs.

Plasmid DNA was extracted from E. coli by using a GeneJET Plasmid Miniprep Kit from Thermo Fisher Scientific. FastDigest restriction endonucleases were purchased from Thermo Fisher Scientific. To get DNA fragments for cloning, PCR was performed with Q5 high-fidelity DNA polymerase from NEB. Other PCR was routinely carried out by using DreamTaq Green PCR Master Mix from Thermo Scientific. Oligonucleotide primers were synthesized in Invitrogen (Grand Island, NY). The sequence and generated restriction sites of the primers are listed in Supplementary Table S1. The PCR products and plasmids digested by restriction endonucleases were recovered from agarose gel after electrophoresis with GeneJET Gel Extraction kit (Thermo Scientific). The competent cells of E. coli were made by CaCl₂ solution and transformed with DNA by fowling the heat-shock procedure. Prior to transformation of Y. lipolytica, the digested DNA fragments were recovered by using DNA Clean & Concentrator kit (Zymo Research, Irvine, CA). Y. lipolytica cell was transformed with linearized plasmid or DNA fragment by using Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA).

FastDigest restriction endonucleases, MuA transposase and T4 DNA ligase were purchased from Thermo Fisher Scientific. DNA Polymerase I, Large (Klenow) Fragment, was purchased from New England Biolabs. All DNA modifying enzymes were used according to the manufacturer’s conditions. All DNA purification procedures were performed according to the manufacturers’ instructions using kits, including GeneJET Plasmid Miniprep kit (Thermo Fischer Scientific) for plasmid extractions from E. coli cells, Zymoclean Gel DNA Recovery kit (Zymo Research) for agarose gel extraction of DNA fragments upon electrophoresis and DNA Clean & Concentrator kit (Zymo Research) for DNA purification and concentration. Bacterial transformations were performed by electroporation using E. cloni 10G ELITE electrocompetent cells (Lucigen).

Paraoxon, 4-nitrophenyl butyrate (4-NPB), 1-naphthyl butyrate (1-NB), 2-naphthyl hexanoate (2-NH) and Fast Red were purchased from Sigma. FastDigest restriction endonucleases, MuA transposase and T4 DNA ligase were purchased from Thermo Fisher Scientific. DNA Polymerase I, Large (Klenow) Fragment, was purchased from New England Biolabs. All DNA modifying enzymes were used according to the manufacturer’s conditions. Oligonucleotides for PCR and adapter cloning experiments (Supplementary Table S17) were from Life Technologies and Sigma-Aldrich. The pGro7 plasmid for GroEL/ES overexpression was obtained from Takara Bio.