| Chapter title |
Expressing Multi-subunit Complexes Using biGBac
|
|---|---|
| Chapter number | 21 |
| Book title |
Protein Complex Assembly
|
| Published in |
Methods in molecular biology, January 2018
|
| DOI | 10.1007/978-1-4939-7759-8_21 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7758-1, 978-1-4939-7759-8
|
| Authors |
Florian Weissmann, Jan-Michael Peters |
| Abstract |
The reconstitution of recombinant protein complexes is facilitated by methods that allow coexpression of their subunits from a single vector. Here we describe a detailed step-by-step protocol for the biGBac cloning method which can be used to generate baculoviral transfer vectors coding for up to 25 subunits of a protein complex (Weissmann et al., Proc Natl Acad Sci U S A 113(19):E2564-E2569, 2016). biGBac is based on Gibson assembly reactions, optimized DNA linker sequences, and uses a hierarchical two-step assembly procedure. In the first assembly step, up to five expression cassettes are combined to generate a polygene cassette. In the second step, up to five polygene cassettes can then be combined to generate transfer vectors coding for up to 25 subunits. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 35 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 9 | 26% |
| Student > Master | 7 | 20% |
| Student > Bachelor | 5 | 14% |
| Student > Ph. D. Student | 3 | 9% |
| Professor | 1 | 3% |
| Other | 2 | 6% |
| Unknown | 8 | 23% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 18 | 51% |
| Chemistry | 3 | 9% |
| Agricultural and Biological Sciences | 3 | 9% |
| Unspecified | 1 | 3% |
| Computer Science | 1 | 3% |
| Other | 0 | 0% |
| Unknown | 9 | 26% |