Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts

Abstract

This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours.

Cell‐free eukaryotic systems for the production, engineering, and modification of scFv antibody fragments

“…Thus, it can be speculated that the presence of the melittin signal sequence at the 5 end of the target gene stabilizes the initiation activity and translational processivity, resulting in significantly increased protein yields. This interpretation is also supported by a publication, in which the authors have shown the stimulatory effect of signal sequences on protein synthesis efficiency in an E. coli-based cell-free protein synthesis system [42]. Interestingly, the stimulatory effect of the signal sequence was mainly dependent on the identity of its second codon.…”

“…Interestingly, the stimulatory effect of the signal sequence was mainly dependent on the identity of its second codon. In particular, in most effective signal sequences, position +2 was occupied by the codon AAA , an observation that also accounts for the melittin signal sequence that was applied in this study . Although melittin‐fused antibody fragments could be produced in sufficient amounts, only very weak binding or no binding at all was detected for these scFv molecules.…”

Beyond oil degradation: enzymatic potential of Alcanivorax to degrade natural and synthetic polyesters

“…Although the enzyme was successfully produced and secreted when it was cloned with its original signal peptide for secretion, best results were obtained when this peptide was replaced by the host's phoA signal peptide ( Supplementary Fig. S3; Ahn et al, 2006). The highest overexpression, assessed by clear zone halos produced by E. coli supernatants on PHB agarose plates, were obtained after 72 h of incubation after inducing with 1 mM of IPTG at 27 C ( Supplementary Fig.…”

Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell‐free system

“…d (+) indicates absorbance greater than to blank control (PNGase treated and untreated, see Figure S2) sample absorbance's for each respective lectin 1-12. Others have reported the comparable yields of EPO with human signal peptide using the E. coli cell-free system (Ahn et al, 2007). (−) indicates absorbance values that are less than blank control (PNGase treated and untreated, see Figure S2) sample absorbance's for each respective lectin 1-12.…”

“…Investigators (Table 1) have used the E. coli CFPS, insect CFPS and CHO CFPS systems for the expression of human EPO (Ahn, Choi, & Kim, 2005;Ahn, Hwang, Lee, Choi, & Kim, 2007;Brodel, Sonnabend, & Kubick, 2014;Brodel, Wustenhagen, & Kubick, 2015;Stech et al, 2014). However, no further optimization studies were reported.…”