Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins

Chloroplastic thioredoxin m functions as a major regulator of Calvin cycle enzymes during photosynthesis in vivo

“…The purified proteins were stored at −80°C (Motohashi et al ., ). Trx m 1, m 2, m 4, x , y 1, y 2, and z were purified as described (Motohashi et al ., , ; Motohashi and Okegawa, ). The concentration of purified proteins was determined with the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA, https://www.thermofisher.com/) using a standard curve of bovine serum albumin concentration.…”

“…The mature forms of Arabidopsis Trx proteins (m1, m2, m4, x, y1, y2, and z) were predicted using Target P 1.1 (Emanuelsson et al, 2000) and expressed as previously described (Motohashi et al, 2009;Motohashi and Okegawa, 2014). The mature form of Trx f1 protein referred to that of recombinant protein by Collin et al (2003).…”

“…The mature form of Trx f1 protein referred to that of recombinant protein by Collin et al (2003). Trx f1 was purified without an affinity tag as described (Motohashi and Okegawa, 2014). The mature form of Trx m3 was purified without an affinity tag as follows: E. coli cells were suspended in 25 mM Tris-HCl (pH 8.1), and disrupted by sonication (Sonifier 250, Branson, Danbury, CT, USA, http://www.emersonin dustrial.com/en-US/branson/Pages/home.aspx) at 4°C.…”

High-throughput recombinant protein expression in Escherichia coli : current status and future perspectives

“…To decrease the propensity by the mRNA around the ribosome binding site to form secondary structures, optimization of the AT-content of N-terminal codons has been demonstrated to be a useful strategy, which was used to promote the overexpression of several proteins from bacteria [75], plants [76] and mammals [77] in E. coli . Moreover, computational tools have been developed to estimate protein expression and design optimal sequences, such as E x E n S o (Expression Enhancer Software) [78], RBS C alculator [79], RBS D esigner [80], UTR D esigner [81] and EMOPEC [82].…”

Evaluation of seamless ligation cloning extract preparation methods from an Escherichia coli laboratory strain

“…We expected that increasing the protein concentration with efficient cell disruption would retain much of the homologous recombination activity in the resulting cell lysates. Sonication and French press are common and efficient methods for protein extraction from E. coli [17][18][19][20][21]. When SLiCE was prepared by sonication in 50 mM Tris-HCl buffer (pH 8.0), the protein concentration was 2.3-fold higher than that extracted by CelLytic B Cell Lysis Reagent ( Fig.…”