Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker

Abstract

Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.

Utilization of a calmodulin lysine methyltransferase co-expression system for the generation of a combinatorial library of post-translationally modified proteins

“…The design, construction, and characterization of high-quality combinatorial libraries [18] of the central linker of mammalian CaM were recently described [17]. Briefly, synthetic gene libraries encoding the native polar and nonpolar (binary patterning) periodicity of the central linker region were designed, synthesized and subcloned into the CaM expression vector, pETCaM1C.…”

“…Briefly, synthetic gene libraries encoding the native polar and nonpolar (binary patterning) periodicity of the central linker region were designed, synthesized and subcloned into the CaM expression vector, pETCaM1C. Individual protein library members lacking the introduction of lysine residues were chosen, transformed into E. coli BL21(DE3) cells, recombinantly expressed in 1L LB-kanamycin media at 37°C, and purified using a TAPP-sepharose affinity resin in a calcium-dependent manner to >95% purity [17]. …”

“…Each individual library member plasmid (pETCaM1C, [17]) was transformed into E. coli BL21(DE3)-star-RIL (Stratagene) competent cells, previously transformed with a modified pET23d expression vector, containing the SUMO-tagged human isoform of CaM KMT [16] and ampicillin resistance. Bacteria were grown in LB media and induced with 1 mM IPTG at 37 °C to simultaneously express the two proteins.…”

“…As an initial step towards using this system as a tool to develop post-translationally modified proteins, we present the application of an E. coli co-expression system to post-translationally trimethylate a recently characterized, high-quality combinatorial library of the mammalian CaM central linker region (residues 68-92) [17]. We show that all tested library members were able to be co-expressed with CaM KMT, modified, and purified.…”

Current Issues in Biowaivers and Biosimilars

“…Platform technologies productively introduce modified changes into a protein structure. They can yield large collections of folded, soluble, highly-expressible, and functionally-novel proteins [14].…”