Evaluation of different promoters and host strains for the high-level expression of collagen-like polymer in Escherichia coli.
The increased expression of collagen-like polymer, CLP3.1-his which consists of 52 repeating peptide (GAPGAPGSQGAPGLQ), in Escherichia coli was investigated. The effects of three promoters, thermally inducible promoter, T7 promoter and T7lac promoter, and three Escherichia coli host strains, BL21, BL21(DE3) and BL21(DE3)[pLysS] which differ in stringency of suppressing basal transcription, were compared. Based on the CLP3.1-his expression level, solubility of CLP3.1-his in cells and basal transcription that occurred in the absence of induction, two expression systems, BL21(DE3) containing plasmid pJY-2 with T7lac promoter and BL21(DE3)[pLysS] containing plasmid pJY-1 with T7 promoter, were selected. With these two expression systems, CLP3.1-his expression levels greater than 40% (g/g) of total cellular proteins and CLP3.1-his concentrations of 0.1-0.2 gl(-1) can be achieved by using Luria-Bertani medium in shake flask batch cultures. The CLP3.1-his accumulated in the cells is totally soluble and no basal transcription was found before induction. These two high-level expression systems are promising for use in scale-up production.
Yin
Jin
J
Biotechnology Process Engineering Center, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Lin
Ju hwa
Jh
Li
Wen tyng
Wt
Wang
Daniel I C
DI
D003001:Cloning, Molecular; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D011108:Polymers / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013045:Species Specificity; D014162:Transfection / Q000379:methods
D011108:Polymers; D011994:Recombinant Proteins; D003094:Collagen