Genes encoding the desired amino acid sequences were purchased from Geneart (Thermo Fisher Scientific) and cloned into the pPICZαB expression vector (Invitrogen). TriFu and CR1(15–17) variants were recombinantly expressed in P. pastoris KM71H using a lab-scale fermenter with small modifications as previously published (32 (link), 34 (link)). Protein purification was accomplished by ion exchange and size exclusion chromatography, similarly to that described previously (32 (link), 34 (link), 49 (link)). In most cases, the N-glycosylation sites were removed by mutating the consensus asparagine to a glutamine residue. For constructs with consensus N-glycosylation sites still present, the endoglycosidase H (Endo Hf, new England Biolabs) was used to deglycosylate the proteins, as described before (34 (link)). Purity and identity of the purified proteins were confirmed by SDS-PAGE analysis similarly as reported before (32 (link)). Aliquots of the engineered complement regulators in PBS were shock frozen in liquid nitrogen and stored at −80 °C until analysis.
Ppiczαb expression vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PPICZαB expression vector is a plasmid designed for the expression of recombinant proteins in Pichia pastoris. It contains the alcohol oxidase 1 (AOX1) promoter for methanol-inducible expression, the Zeocin resistance gene for selection, and the α-factor signal sequence for secretion of the expressed protein.
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Lab products found in correlation
2 protocols using ppiczαb expression vector
1
Recombinant Complement Regulator Expression
2
Heterologous Expression of OGOX1.2 in Pichia
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The gene encoding the mature OGOX1.2 isoform from Arabidopsis thaliana (AT4G20830.2) was fused downstream of the SUMOstar sequence developed by LifeSensor Inc. (https://lifesensors.com/ ) that also included the sequences encoding the FLAG- (DYKDDDDK) and 6xHis-tags (HHHHHH) (https://lifesensors.com/wp-content/uploads/2019/09/2160_2161_Pichia_SUMOstar_Manual-1.pdf ). The sequence of the chimeric gene, here referred to as FHS-OGOX1, was codon-optimized with the codon usage of Pichia pastoris by using the online tool OPTIMIZER (http://genomes.urv.es/OPTIMIZER/ )26 (link) and was entirely synthesized by Genescript (https://www.genscript.com/ ) by adding the bases of the restriction sites PstI and XbaI at the 5I and 3I ends, respectively, of the gene. The gene was then cloned in pPICZαB expression vector (Invitrogen, San Diego, USA) in frame with the sequence encoding the yeast α factor for the secretion of recombinant proteins in the medium.
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