GMP-Creative Biolabs provides one-stop customer-oriented services including biopharmaceutical contract R&D, process development, cGMP manufacturing, QA & QC solutions for scientific research, preclinical, clinical and commercial supply. We proceed your projects according to your specific demands in quality, timeline and budget, supporting researchers across different therapeutic areas. Clear, open and regular communication with customers is as important to our success as our in-depth scientific knowledge. more
Creative Biolabs provides Escherichia coli (E.coli) as host to produce recombinant proteins. With no doubt that E.coli is the most widely utilized host for heterologous protein expression. It dominates the bacterial expression systems. For laboratory investigations and initial development in commercial activities, E. coli is the preferred system. The ability that E. coli now can produce mg level of glycosylated proteins promises E. coli broaden applications in the future.
Figure: Anatomy of an expression vector. (Front Microbiol. 2014)
As one of the most popular expression platforms, E. coli is the host cell to produce human insulin, the first recombinant protein, which was approved for marketing in 1982. E. coli has many well-known advantages to provide a mean for economical and rapid production of recombinant proteins. Firstly, E. coli has unparalleled fast growth kinetics. In optimal growing conditions, its doubling time is about 20 mins which means it may reach stationary phase in a few hours. Secondly, it is easy for E. coli to achieve high cell density cultures. Thirdly, the media of E. coli culture is readily available and inexpensive. Finally, plasmid transformation of E. coli is fast and easy, which can be performed in as little as 5 mins. In traditional methods, E. coli is used to produce non-glycosylated proteins. The discovery that N-linked glycosylation system exists in a Gram-negative bacterium Campylobacter jejuni and can be transferred to E. coli makes it possible to use E. coli to generate glycoproteins. Protein folding is an important issue in E. coli system. Heterologous protein accumulation often occurs in inclusion bodies. And proper folded proteins can be obtained from inclusion bodies via controlling aggregation. Cytoplasm folding and periplasm folding are often desirable to obtain soluble and bioactive proteins without refolding. According to various researches, the cytoplasmic space is promising for the production of Fab antibody fragments. And a number of secreted, disulfide-bond-containing proteins fold readily in the periplasm including single-chain Fv antibody fragments and human growth hormone. Whatever proteins needed to produce, the following factors are taken into consideration to develop stable cell lines.
|a.||E. coli strains: BL21 (DE3) and some derivatives of the K-12 are the most popular strains. BL21 is derived from the B line via various modifications. BL21 cells have many genetic characteristics. They are deficient in the Lon protease, OmpT and have hsdSB mutation. These modifications facilitate expressed proteins retain intact and prevent plasmid losing. In the popular BL21 (DE3) strain, the λDE3 prophage was inserted in the chromosome of BL21 and contains the T7 RNAP gene under the lacUV5 promoter. K-12 strains lack of all known E. coli virulence genes, which makes them prototypes of safe and nonpathogenic bacterial strains. Now the widely used strains from the K-12 repertoire include AD494, OrigamiTM and HMS174, which have genetic difference. Moreover, they have their λDE3 derivative and T7 RNA system can be used.|
|b.||Vectors: the optimized expression vectors are the result of multiple combinations of replicons, promoters, multiple cloning sites, selection markers, etc. The major features of an expression vectors are shown in the figure 1: Origin (pMB1, ColE1, pUC, etc.), promoter (lac/lac UV5, tac/trc, T7/T7/lac, araPBAD, etc.), affinity tags (peptide tags or fusion partners), coding sequence for tag removal (enterokinase, thrombin, factor Xa, TEV) and selection marker (antibiotic resistance or plasmid addiction systems).|
Creative Biolabs provides the following microbial systems for various proteins expression:
To discuss your Escherichia coli (E. coli) Cell Lines demands or to request a proposal, please contact us by
For R&D use only, not directly for clinical use.