Selection of K. lactis cells transformed with pKLAC-series vectors by growth on YCB Agar Medium containing 5 mM acetamide nearly completley enriches transformant populations for cells that have inserted multiple tandem copies of the linear vector into the genome. Highly efficient multicopy integration can be exploited to rapidly create strains expressing multiple heterologous proteins using a single round of transformation and selection(3). This is accmplished by co-transforming K. lactis cells with two or more pKLAC-series vectors, each containing a different heterologous gene. Successful expression of up to four proteins has been reported using this method (3), although co-expression of more proteins may be possible. Read et al., have describe this method in detail (3), however, an abbreviated protocol is provided here.
- Separately digest 2 μg of each pKLAC2 vector contaning a gene of interest with 20 units of SacII in 50 μl of 1X NEBuffer 4 (supplied as a 10X stock) at 37oC for 2 hours.
For example, to produce to proteins (protein A and protein B), the two constructs pKLAC2-gene-A and pKLAC2-gene-B would each be digested. The procedure can be performed with any combination of pKLAC-series vectors.
- Pool the restriction digests and desalt the digested vectors using a commercially available DNA fragment purification kit (e.g., Qiagen's QIAquick™ PCR Purification Kit). Elute in 30 μl of deionized water.
A total of 1 μg of each linearized vector in a volume less than 15 μl will be needed to transform K. lactis cells. DNA may be stored frozen at -20oC for up to one month prior to transforming K. lactis cells.
- Transform K. lactis GG799 cells with 15 μl of linearized vector DNA (contaning 1 μg of each vector) using the method for "Transformation of K. lactis GG799 cells."
It is important to note that not all transformants will express both proteins. Acetamide selection nearly completley enriches transformant populations for multicopy integrants, however, it does not select for each vector individually. Therefore, it is important to screen multiple transformants for co-expression of proteins. In one study (3), 70-93% of strains transformed with two expression vectors produced both heterologous proteins, and 63% of cells transformed with three vectors produced all three proteins.