Presentation Information
[P04-593]Production of recombinant spider silk protein in Escherichia coli
○Chung-Jen Chiang1, Yun-Peng Chao2 (1. Department of Medical Laboratory Science and Biotechnology, China Medical University (Taiwan), 2. Department of Chemical Engineering, Feng Chia University (Taiwan))
Keywords:
spider silk protein,spidroin,expression system
[Purpose]Dragline spider silk protein (spidroin) is a natural fiber with the strongest composite properties, known for its extremely high tensile strength, elasticity, and toughness. It has high potential applications ranging from textile fibers to monofilament surgical sutures. The techno-economic analysis indicates that producing recombinant spider silk proteins in Escherichia coli is economically viable. However, the technical challenge remains because spidroins are highly repetitive proteins with multiple tandem repeats of sequences rich in glycine and alanine. Adverse events, including genetic instability and metabolic burden, usually occur when recombinant spidroins are overexpressed in E. coli. Accordingly, the study aimed to develop an expression system to address this issue.
[Method]The pET-32a plasmid was engineered to include the tetO2 site and replace lacI with tetR. This was achieved by PCR with synthetic primers. Consequently, the gene construct produced the pTet-32a plasmid. Meanwhile, the coding sequence of Nephila clavipes MaSp1 was synthesized and flanked by NheI and SpeI restriction sites. The recursive directional genetic assembly was applied to obtain 16-mer and 32-mer MaSp1. The subcloning of 16-mer and 32-mer MaSp1 into the pET-32a and pTet-32a plasmids resulted in the pSilk16 and pSilk32 plasmids, respectively. To produce MaSp1, the E. coli strain (N30G) was engineered with the enhanced glyV tRNA.
[Results]Two MaSp1 coding sequences were synthesized and expressed in the N30G strain. The strains carrying the MaSp1 clone were cultured with glycerol and induced for protein production. Consequently, the strain could produce the 16-mer MaSp1 with a more balanced codon usage. The E. coli BL21 (DE3) strain and the pET plasmids are most commonly employed for protein expression. However, this system is afflicted with high basal expression, which causes cell toxicity and induces mutations in the expressed gene. This issue was addressed by replacing the lacI-lacO element on the pET plasmid with the tetR-tetO2 cassette. The toxic IPTG inducer was also eliminated by using the N30G strain, which harbors the L-arabinose-regulated T7 gene 1. Finally, the 32-mer MaSp1 was highly expressed in the N30G strain carrying the pSilk32 plasmid. The strain's growth was marginally affected, and the plasmid could be stably maintained in the cell.
[Consideration]Our result suggests that an overemphasis on preferred codons of the cloned gene may disrupt the balance between codon usage rates and tRNA levels, thereby reducing translation efficiency. The tetR-tetO2 set-up significantly enhances stringent regulation of the T7 expression system. This widens the regulatory window in favor of producing hard-to-express proteins.
[Conclusion]This study developed a novel expression system with high stringency and inducibility. It may hold promise for producing recombinant spidroins with high MW. This work is currently underway in our lab.
[Method]The pET-32a plasmid was engineered to include the tetO2 site and replace lacI with tetR. This was achieved by PCR with synthetic primers. Consequently, the gene construct produced the pTet-32a plasmid. Meanwhile, the coding sequence of Nephila clavipes MaSp1 was synthesized and flanked by NheI and SpeI restriction sites. The recursive directional genetic assembly was applied to obtain 16-mer and 32-mer MaSp1. The subcloning of 16-mer and 32-mer MaSp1 into the pET-32a and pTet-32a plasmids resulted in the pSilk16 and pSilk32 plasmids, respectively. To produce MaSp1, the E. coli strain (N30G) was engineered with the enhanced glyV tRNA.
[Results]Two MaSp1 coding sequences were synthesized and expressed in the N30G strain. The strains carrying the MaSp1 clone were cultured with glycerol and induced for protein production. Consequently, the strain could produce the 16-mer MaSp1 with a more balanced codon usage. The E. coli BL21 (DE3) strain and the pET plasmids are most commonly employed for protein expression. However, this system is afflicted with high basal expression, which causes cell toxicity and induces mutations in the expressed gene. This issue was addressed by replacing the lacI-lacO element on the pET plasmid with the tetR-tetO2 cassette. The toxic IPTG inducer was also eliminated by using the N30G strain, which harbors the L-arabinose-regulated T7 gene 1. Finally, the 32-mer MaSp1 was highly expressed in the N30G strain carrying the pSilk32 plasmid. The strain's growth was marginally affected, and the plasmid could be stably maintained in the cell.
[Consideration]Our result suggests that an overemphasis on preferred codons of the cloned gene may disrupt the balance between codon usage rates and tRNA levels, thereby reducing translation efficiency. The tetR-tetO2 set-up significantly enhances stringent regulation of the T7 expression system. This widens the regulatory window in favor of producing hard-to-express proteins.
[Conclusion]This study developed a novel expression system with high stringency and inducibility. It may hold promise for producing recombinant spidroins with high MW. This work is currently underway in our lab.
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