A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts

Abstract

We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.

Robust Recombinant Expression of Human Placental Ribonuclease Inhibitor in Insect Cells

“…The vector design for the recombinant expression of RI in insect cells is based on two previous vector systems: the Profinity Exact system [ 19 ], which utilizes an immobilized, engineered, fluoride triggered subtilisin [ 20 ] that both recognizes and avidly binds to the small N-terminal co-expressed affinity tag in a protein fusion; and pONE [ 21 ] in which the baculovirus transfer vector (pONE30A) is optimized to yield high protein quantity as a fusion protein. An insect cell codon optimized version of the fusion section of pPal8 was introduced to pONE30A at NdeI/NotI sites to create a new baculovirus transfer vector named pOPal30 compatible with the original Profinity Exact system.…”

Ligand-induced conformational rearrangements regulate the switch between membrane-proximal and distal functions of Rho kinase 2

“…These proteins are huge (2 × 1354 and 2 × 1388 aa for ROCK1 and ROCK2, respectively), and hard to express in heterologous recombinant systems. Using our recently developed unified expression system (pONE) 31 , we successfully expressed the full-size, dimeric ROCK2 protein with an MBP tag in insect cells, in sufficient amounts required for structural studies.…”

“…Protein expression and purification ROCK2, ROCK2-KD constructs, expression, and purification. The synthetic gene of human ROCK2 (1-1388) was cloned into pONE30A 31 vector between AvrII and NotI cleavage sites. The ROCK2 kinase domain (1-420) construct (ROCK2-KD) truncation was prepared using primers 5′-GATCGCTAGCCCTAGGGGATCC-3′ and 5′-GATCGCGGCCGCTTCGCGGCAGGAGG-3′, cloned between NheI and NotI restriction sites.…”

“…RhoA construct, expression, and purification. The synthetic gene of the human RhoA (1-181) was cloned into a pONE10K vector 31 between NcoI-NotI cleavage sites. The construct was transformed into Rosetta2 E. coli cells and the protein (containing additional AS-sequence at the N-terminus and the -AAHHHHHH sequence at the C-terminus) was expressed at 37°C in the presence of 50 µg/mL kanamycin and 30 µg/mL chloramphenicol.…”

Beta-Secretase 1 Recruits Amyloid-Beta Precursor Protein to ROCK2 Kinase, Resulting in Erroneous Phosphorylation and Beta-Amyloid Plaque Formation

“…The synthetic gene (Geneart AG, Regensburg, Germany) of human APP646-695 was cloned into the pONE10K [49] vector between NheI and NotI cleavage sites, using primers 5′-GGGCTAGCGTGATGCTGAAGAAGAAACAG-3′ and 5′-GGGCGGCCGCCTAG-CAGTTCTGCATCTGCTCAAA-3′. The APP646-664 construct was prepared using primers…”