ISSN:
0006-3592
Keywords:
lambda phage
;
lysogeny
;
lytic state
;
partial lysis
;
multiplicity of infection
;
temperature induction
;
intracellular
;
extracellular product
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
The integrated state of λ in the host chromosome in lysogeny can be combined with its extrachromosomal replication in the lytic state to achieve high cloned gene productivities. Our previous studies on λ expression systems21,22 have shown 100% segregational stability of the cloned gene in lysogeny and cloned gene product levels up to 15% of total cell protein in a mutant lytic state. However, the expression phase of systems based on Escherichia coli JM109 and JM105 showed partial lysis of the productive culture despite a mutation in the lysis gene S of the lambda vector resulting in extracellular release of the cloned gene product. In the current study, we have eliminated partial lysis in the expression phase of λ systems and conducted a detailed comparative analysis of these systems in relation to maximization of cloned gene productivity. The elimination of partial cell lysis by using a nonpermissive strain Y1089 did not enhance product yields vs. earlier systems that exhibited partial lysis. The elimination of nonessential λ protein production by construction of a new vector NP326 did not yield higher product yields presumably because of the small fraction of these proteins in the lytic state. Temperature induction of the lysogen Y1089(NM1070) resulted in higher product levels than direct infection of Y1089 by the phage vector at a high multiplicity. Using infection experiments, we found the promoter lacUV5 in the vector λZEQS to yield threefold higher product levels than lac in NM1070, suggesting possible further enhancement of productivity with stronger promoters. The occurrence or absence of partial lysis in λ systems could be used beneficially to achieve extracellular or intracellular product as desired. The large capacity of λ vectors for insert DNA suggests potential applications in obtaining highly amplified levels of operons and multienzyme systems. © 1992 John Wiley & Sons, Inc.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bit.260400608
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