Periplasmic expression of human growth hormone via plasmid vectors containing the lambdaPL promoter: use of HPLC for product quantification.
The influence of different factors acting on Escherichia coli periplasmic expression of recombinant human growth hormone (hGH) in shake flask cultures has been investigated. Bacterial vectors containing the phage lambdaP(L) promoter, which is temperature activated, were utilized. Four different signal peptides were compared: DsbA, npr, STII and one derived from the natural hGH signal peptide, this last used as a reference. Other factors such as medium composition, optimized induction and expression conditions, and different bacterial strains were also studied. The determination of hGH, carried out directly in osmotic shock fluids, was based on an isocratic reversed-phase high-performance liquid chromatography method, which allows direct, rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space immediately after or even during fermentation. The level of hGH production increased 2.5-fold compared with the reference vector, reaching a level of 3.9 +/- 0.63 micro g/ml/A(600) (n = 6; coefficient of variation = 16.2%). The expression level was affected by the signal peptide and by the induction conditions, being more effective when activation started in the early logarithmic phase which, however, exhibited remarkably different optical density (OD) according to medium composition. Our results thus indicate that 6 h activation at 40-42 degrees C, starting with an OD (A(600)) of approximately 3 in a very rich medium, were conditions capable of providing the maximum secretion level for a vector utilizing the DsbA signal sequence and E.coli W3110 or RB791 as host cells.
Biotechnology Department, IPEN-CNEN, Av. Prof. Lineu Prestes, 2242, Cidade Universitária, 05508-900, São Paulo, Brazil.
Carlos R J
CR
Soares
Fernanda I C
FI
Gomide
Eric K M
EK
Ueda
Paolo
P
Bartolini
D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005822:Genetic Vectors; D019382:Human Growth Hormone / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019897:Periplasm / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D013696:Temperature; D013997:Time Factors; D014764:Viral Proteins / Q000235:genetics*
C024013:DNA replication complex protein, Bacteriophage lambda; D021382:Protein Sorting Signals; D014764:Viral Proteins; D019382:Human Growth Hormone