pOPIN-GG: A resource for modular assembly in protein expression vectors

Abstract

The ability to recombinantly produce target proteins is essential to many biochemical, structural, and biophysical assays that allow for interrogation of molecular mechanisms behind protein function. Purification and solubility tags are routinely used to maximise the yield and ease of protein expression and purification from E. coli. A major hurdle in high-throughput protein expression trials is the cloning required to produce multiple constructs with different solubility tags. Here we report a modification of the well-established pOPIN expression vector suite to be compatible with modular cloning via Type IIS restriction enzymes. This allows users to rapidly generate multiple constructs with any desired tag, introducing modularity in the system and delivering compatibility with other modular cloning vector systems, for example streamlining the process of moving between expression hosts. We demonstrate these constructs maintain the expression capability of the original pOPIN vector suite and can also be used to efficiently express and purify protein complexes, making these vectors an excellent resource for high-throughput protein expression trials.

The stem rust effector protein AvrSr50 escapes Sr50 recognition by a substitution in a single surface‐exposed residue

“…For protein expression and purification, the cDNA of three AvrSr50 variants, AvrSr50 WT , AvrSr50 RKQQC and avrSr50 QCMJC (Chen et al ., 2017 ) from this point forwards referred to as AvrSr50‐A1, AvrSr50‐C and avrSr50‐B6, respectively, were PCR‐amplified, excluding their signal peptide as identified by SignalP‐5.0 (Almagro Armenteros et al ., 2019 ), and cloned into a modified, Golden Gate‐compatible, pOPIN expression vector using Golden Gate cloning (Supporting Information Table S1 , Bentham et al ., 2021 ). Primers consisted of a gene‐specific sequence flanked by overhangs compatible for Golden Gate cloning (Table S2 ) and Golden Gate digestion/ligation reactions and cycling were carried out as described previously (Iverson et al ., 2016 ).…”

The structural repertoire ofFusarium oxysporumf. sp.lycopersicieffectors revealed by experimental and computational studies

“…2C). Over half of these were found in Ascomycetes (73), with expanded families in species of Colletotrichum, Diversisora, and ), as well as many formae speciales of Fusarium oxysporum and other Fusarium species (S2 Table ). Expanded families of FOLD proteins were observed in the genus of Glomeromycota that form arbuscular mycorrhiza in plant roots, while two putative FOLD effectors were also predicted in the ectomycorrhizal fungus Piloderma olivaceum (basidiomycete), which forms mutualistic associations with conifer and hardwood species [44].…”

“…All of the primers were synthesised by IDT (Coralville, Iowa, USA) (S5 Table ). All genes were cloned into a modified, Golden-Gate-compatible, pOPIN expression vector [73]. The final expression constructs contained N-terminal 6xHis-GB1-tags followed by 3C protease recognition sites.…”

Allelic compatibility in plant immune receptors facilitates engineering of new effector recognition specificities

“…The RGA5, APB and Pikm-1 HMA domains as well as the AVR-Pik effector variants and AVR-Pia effector were cloned into pOPIN-GG vector pPGN-C (Bentham et al, 2021) with a cleavable N-terminal 6xHIS-GB1-3C tag via Golden Gate cloning with BsaI . AVR-Pii effector domain was cloned with a cleavable N-terminal MBP tag and an uncleavable C-terminal 6xHIS via In-fusion cloning into pOPINE (Berrow et al, 2007).…”

“…AVR-Pii effector domain was cloned with a cleavable N-terminal MBP tag and an uncleavable C-terminal 6xHIS via In-fusion cloning into pOPINE (Berrow et al, 2007). For co-expression with the APB HMA for crystallography studies, AVR-PikF was cloned into pPGC-K (Bentham et al, 2021) without a tag via Golden Gate cloning with BsaI .…”