Enhanced secretion of adhesive recognition sequence containing hirudin III mutein in E. coli.
It has been previously shown that Escherichia coli L-asparaginase II (L-ASP) signal peptide is capable of being utilized to direct extracellular secretion of hirudin III (HV3) in shake flask. In this study HV3 muteins R33G34D35(S36)-HV3 were generated by introduction of adhesive recognition sequence RGD(S) into the non-functional region of HV3. The resultant recombinants were cultivated on 30 l bioreactor scale using L-ASP signal peptide expression system and the optimized fed-batch cultivation was well established. After cultivation for approximately 11 h the secreted product accumulated up to approximately 1 g l(-1), which means 17-fold increase in productivity compared to initial expression in shake flask. N-terminal analysis, pI measurement, and MALDI mass spectral analysis on mutein R33G34D35S36-HV3 confirmed the authenticity of the product. Compared to wild-type HV3 and R33G34D35HV3, the mutein R33G34D35S36-HV3 exhibits the improved pharmacological activity. Collectively, a novel secretion strategy using L-ASP signal peptide for the rapid, efficient and cost-effective production of HV3 mutein possessing improved pharmacological activity on bioreactor scale has been well established. Using this expression system downstream processing becomes very simple because secreted product is mature, soluble, active, and without N-terminal extension of Met, which is quite critical for most therapeutic protein to reduce the side effect in clinic use. Thus, it provides a promising alternative for extracellular production of other difficult-to-express protein for biopharmaceutical use.
Tan
Shuhua
S
School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, P.R. China. tohike@hotmail.com
Wu
Wutong
W
Li
Xiangyu
X
Cui
Li
L
Li
Bing
B
Ruan
Qiping
Q
D000925:Anticoagulants / Q000378:metabolism; D019149:Bioreactors; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D006629:Hirudins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007526:Isoelectric Point; D050505:Mutant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization