MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

Abstract

Background: The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms.

Results: We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented.

Conclusions: The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.

Figure 1

Comparison of protein expression screening success. The percentage of success for each step in the protein production pipeline is shown. The number of targets represented at each stage is written above the bars.

Figure 2

Assessing soluble and insoluble protein expression levels in the medium throughput strategy. a. SDS-PAGE of Insoluble (I) and soluble (S) crude cellular extracts of the E. coli expression cultures. Targets 50 to 60 are shown. Black circles surround bands of correct size for a given target. b. Western-blot using anti-His antibody of selected targets highlighted after purification of soluble cell lysis extracts on mini-affinity (His) columns. Selected targets between 156 and 181 are shown. Pink circles highlight bands of correct size for a given target.

Figure 3

Up-scale and purification of targets from P. abyssi. a. SDS-PAGE gel of up-scaled and purified HIS-tagged targets F75, F124, F127 and F167 before and after final polishing on Co²⁺ specific beads (SN = Supernatant of cell culture; B = after incubation with Dynabeads TALON; M = protein markers; F75 = test line; NI = F75 without induction). b. 12% SDS-PAGE analysis of a P. abyssi RadA (PAB0164, target 92 round R1) pull-down experiment. Proteins identified using an Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics) are indicated with arrows.

Figure 4

Up-scale purification and crystallization of R-Z3597 from Zobellia galactanivorans. a. SDS-PAGE gel of up-scaled and purified His-tagged target Zg3597. Lane 1, soluble extract; lane 2, purified R-Z3597 by affinity chromatography using a Ni²⁺-charged resin; lane 3, R-Z3597 purified by size exclusion gel chromatography; M, standard molecular weight markers. b. Orthorhombic crystals of Zg3597 (0.1 × 0.1 × 0.5 μm³)

Figure 5

Up-scale purification and crystallization of Stanniocalcin 1 of Sparus aurata. a. SDS-PAGE gel of up-scaled and purified His-tagged target STCA1. b. First plate-like crystals of STCA1 (approximate size of 0.05 × 0.05 × 0.008 μm³).

Figure 6

Comparison of soluble and insoluble protein expression percentage in the three-domain system. Global comparison of the successful expression obtained from archaea, bacteria and eukarya. Eukarya are represented by the blue bars, Bacteria are represented by the red bars and Archaea are represented by the yellow bars.