کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9886104 | 1537495 | 2005 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage
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کلمات کلیدی
mAbdhps - DPSMonoclonal antibody - آنتی بادی مونوکلونالCysteine residue - باقی مانده سستیونProtein expression - بیان پروتئینdihydropteroate synthetase - دی هیدروپرتات سینتاتازDihydropteroate synthase - دی هیدروپروتئات سنتازProtein engineering - مهندسی پروتئینPhage display - نمایش فاژیwild type - نوع وحشی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - General Subjects - Volume 1724, Issues 1â2, 20 June 2005, Pages 146-154
Journal: Biochimica et Biophysica Acta (BBA) - General Subjects - Volume 1724, Issues 1â2, 20 June 2005, Pages 146-154
نویسندگان
Eeva-Christine Brockmann, Urpo Lamminmäki, Petri Saviranta,