Abstract
Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.
Highlights
Since the mammalian hormone somatostatin was first used to realize heterologous expression in Escherichia coli (Itakura et al, 1977), the expression of recombinant protein has developed rapidly for various applications, including industrial enzyme production (Headon and Walsh, 1994), biopharmaceuticals (Jozala et al, 2016), and vaccine production (Christodoulides et al, 2001)
The pcaHG98 gene was amplified by PCR with primers pcahg9801 and pcahg9802 (Figure 1) using strain NCIMB 9866 gDNA or bacteria lysate as a template
Protocatechuate 3,4-dioxygenase catalyzes the cleavage of PCA into β-carboxycis, cis-muconate (Harwood and Parales, 1996). pcaHG98 encodes a putative protocatechuate 3,4-dioxygenase in NCIMB 9866
Summary
Since the mammalian hormone somatostatin was first used to realize heterologous expression in Escherichia coli (Itakura et al, 1977), the expression of recombinant protein has developed rapidly for various applications, including industrial enzyme production (Headon and Walsh, 1994), biopharmaceuticals (Jozala et al, 2016), and vaccine production (Christodoulides et al, 2001). There are some short peptide tags for E. coli expression systems wherein the amino acid sequences are generally 15 residues or less (Ki and Pack, 2020), including the Arg-tag (Sassenfeld and Brewer, 1984), FLAG-tag (Hopp et al, 1988), His-tag (Hochuli et al, 1987), c-myc-tag (Ferrando et al, 2001), S-tag (Karpeisky et al, 1994), Strep-tags (Schmidt and Skerra, 1993), and Fh8 and H tags (Costa et al, 2013) These peptide tags can improve the solubility of a target protein by regulating the process of protein transcription and translation (Ki and Pack, 2020). The identification of additional fusion protein and peptide tags is conducive to finding more beneficial common features for protein heterogeneous expression
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