Development of an expression system for eukarytoic proteins in methylotropic bacteria Page: 1 of 4
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LA- SVu-- 96-- /03
FINAL REPORT R ECFIVED
SUBCONTRACT NO. 9-XH3-4312J-1 SEP 23 3U9g
PI: Mary E. Lidstrom 4
Institution: California Institute of Technology
Title: Development of an Expression System for Eukarytoic Proteins in Methylotrophic
The objective of this project was to develop an expression vector for methylotrophic
bacteria for use in the production of 13C- and 2H-labelled eukaryotic proteins by growing
methylotrophic bacteria on labelled methanol or methylamine. The eukaryotic proteins
calmodulin and troponin C were chosen as test cases. Genes encoding both proteins were
cloned into different constructions and tested for expression. Moderate amounts of
troponin C were found with one of the constructions.
1. Construction of a Eukaryotic Expression Vector for Methylotrophs
A cloning vector that replicates in a variety of methylotrophic bacteria, the broad-
host-range vector pECT5, was used for the initial constructions of the expression vectors.
It contained an IncQ replicon, a streptomycin-resistance gene for selection, a polylinker to
facilitate cloning, and a tac promoter for expression. A second vector was also generated,
containing the mau promoter, for methylamine-regulated expression.
2. Demonstration of the Expression System
Clones containing the gene for either chicken calmodulin or avian troponin C were
provided by Cliff Unkefer's group, and appropriate restriction fragments each containing
the entire gene were subcloned into both types of expression vectors. These clones were
confirmed to be correct, and then were transferred into three different methylotrophic
bacteria, Methylophilus methylotrophus AS 1, Methylobacteriumflagellatum KT, and
Methylobacterium extorquens AML. Each strain was grown under various conditions in
attempts to optimize expression, and cells were tested for production of either calmodulin
or troponin C. Production of these proteins was assessed by screening the cells for
appearance of new polypeptides of the correct size on SDS-PAGE gels. No evidence was
obtained for expressin of troponin C, but it appeared that calmodulin was expressed in two
strains, Methylophilus methylotrophus ASl and Methylobacteriumflagellatum KT
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Lidstrom, M.E. Development of an expression system for eukarytoic proteins in methylotropic bacteria, report, September 1, 1996; New Mexico. (https://digital.library.unt.edu/ark:/67531/metadc675212/m1/1/: accessed March 23, 2019), University of North Texas Libraries, Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.