Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: temperature plays a major role in protein yields.
When producing recombinant protein for therapy, it is desirable not only to obtain substantial amounts of the protein, but also to make sure that potential contaminants such as inducing agents are not present in the final product. To prevent this, one can use expression systems in which the promoter (lambdaP(L)) is activated by a temperature shift that denatures a repressor (e.g., cIts). In this manner, hGH was successfully expressed and secreted in Escherichia coli periplasm, with specific yields well above 1 microg ml(-1) A(600)(-1), after a temperature shift from 30 to 42 degrees C. However, attempts to express a related hormone, human prolactin, employing the same protocol were unsuccessful, providing 0.03 microg ml(-1) A(600)(-1) at the most. A process is described in which this labile protein is obtained from a cIts(-) strain under optimized temperature condition (37 degrees C). The highest periplasmic secretions of prolactin ever reported were thus obtained: 0.92+/-0.10 microg ml(-1) A(600)(-1) at an optical density of approximately 3 A(600) units in shake flask cultures and approximately 1 microg ml(-1) A(600)(-1), at an OD of 35 A(600) units, via a rapid and flexible batch feed process in laboratory bioreactor. Purified hPRL was monomeric, correctly processed (Mr=22,906), properly folded and bioactive (51.5+/-24.1 IU mg(-1)).
Soares
C R J
CR
Biotechnology Department, IPEN-CNEN, Av. Lineu Prestes, 2242 Cidade Universitária, 05508-900 São Paulo, Brazil. crsoares@ipen.br
Ueda
E K M
EK
Oliveira
T L
TL
Gomide
F I C
FI
Heller
S R
SR
Bartolini
P
P
D010582:Bacteriophage lambda / Q000235:genetics*; D018929:Cell Culture Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D013006:Growth Hormone / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D011388:Prolactin / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D011388:Prolactin; D013006:Growth Hormone