Applications of recombinant DNA technology to protein analysis have been demonstrated using the trimethoprim resistant (Tp$\sp{\rm R}$)R388 type II dihydrofolate reductase (DHFR).
DHFR fusion vectors were constructed which contain cloning sites in the C-terminal coding region of the DHFR. Segments of nematode major sperm protein (MSP) fused to the DHFR were recognized by antibody to MSP. A 15 aa segment of MSP sequence, fused to the DHFR, was shown to be sufficient to elicit antibody to MSP. A two-step procedure was developed for purifying the DHFR proteins.
A selectable FokI/supF cassette was developed to facilitate DNA excision and replacement mutagenesis. When the cassette is inserted into DNA, the presence of the tyrosine tRNA suppressor gene (supF) contained on the cassette is selected by the suppression of amber mutations in the recipient host. Subsequent refined mutagenesis is possible due to the unique cleavage properties of FokI. The cassette/vector system was used to produce a deletion corresponding to amino acid residues 2-7 of the DHFR which did not noticeably impair Tp$\sp{\rm R}$. Resistance was abolished, however, by a deletion of amino acid residues 2-21.
A structural gene was synthesized which contains many unique restriction sites and encodes a Tp$\sp{\rm R}$ DHFR which is 10 aa shorter at the N-terminus relative to natural type II DHFRs. High copy number vectors which utilize strong promoters to transcribe the dhfr gene and the primer for plasmid replication were constructed to overproduce the mini DHFR and a full-length derivative.
Site-directed mutagenesis was used to test the importance of glu-58 and thr-48 in a putative folate binding site of the mini and full-length DHFRs. The introduction of thr-58 and/or glu-48 destroyed in vivo function of the DHFRs. Tp$\sp{\rm R}$ was retained in the full length gln-58 R388 DHFR, but not in the mini gln-58 DHFR. Through random mutagenesis a mini Tp$\sp{\rm R}$gln-58 R388 DHFR was obtained that contained a duplication of leu-pro-ser, at the N-terminus. Successive additions of the leu-pro-ser triplet to the N-terminus appear to stabilize the functional form of the mini DHFR.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16196 |
| Date | January 1988 |
| Creators | Vermersch, Polly Smith |
| Contributors | Bennett, George N. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 241 p., application/pdf |
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