PMID- 17827531
OWN - NLM
STAT- MEDLINE
DCOM- 20071003
LR  - 20191110
IS  - 1073-6085 (Print)
IS  - 1073-6085 (Linking)
VI  - 36
IP  - 1
DP  - 2007 May
TI  - Enhanced secretion of adhesive recognition sequence containing hirudin III mutein 
      in E. coli.
PG  - 1-8
AB  - 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.
FAU - Tan, Shuhua
AU  - Tan S
AD  - School of Life Science and Technology, China Pharmaceutical University, Nanjing, 
      210009, P.R. China. tohike@hotmail.com
FAU - Wu, Wutong
AU  - Wu W
FAU - Li, Xiangyu
AU  - Li X
FAU - Cui, Li
AU  - Cui L
FAU - Li, Bing
AU  - Li B
FAU - Ruan, Qiping
AU  - Ruan Q
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Switzerland
TA  - Mol Biotechnol
JT  - Molecular biotechnology
JID - 9423533
RN  - 0 (Anticoagulants)
RN  - 0 (Hirudins)
RN  - 0 (Mutant Proteins)
RN  - 0 (Platelet Aggregation Inhibitors)
RN  - 0 (Protein Sorting Signals)
RN  - 0 (Recombinant Proteins)
SB  - IM
MH  - Anticoagulants/metabolism
MH  - Bioreactors
MH  - Chromatography, High Pressure Liquid
MH  - Escherichia coli/growth & development/*metabolism
MH  - Hirudins/*chemistry/isolation & purification/*metabolism
MH  - Isoelectric Point
MH  - Mutant Proteins/*chemistry/isolation & purification/*metabolism
MH  - Platelet Aggregation Inhibitors/metabolism
MH  - *Protein Sorting Signals
MH  - Recombinant Proteins/chemistry/isolation & purification/metabolism
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
EDAT- 2007/09/11 09:00
MHDA- 2007/10/04 09:00
CRDT- 2007/09/11 09:00
PHST- 2006/08/08 00:00 [received]
PHST- 2006/11/07 00:00 [revised]
PHST- 1999/11/30 00:00 [accepted]
PHST- 2007/09/11 09:00 [pubmed]
PHST- 2007/10/04 09:00 [medline]
PHST- 2007/09/11 09:00 [entrez]
AID - MB:36:1:1 [pii]
AID - 10.1007/s12033-007-0002-8 [doi]
PST - ppublish
SO  - Mol Biotechnol. 2007 May;36(1):1-8. doi: 10.1007/s12033-007-0002-8.