Flow Cytometric Screening of cDNA Expression Libraries for ...

Biotechnol. Prog. 2004, 20, 963−967

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Flow Cytometric Screening of cDNA Expression Libraries for Fluorescent Proteins Paul H. Bessette and Patrick S. Daugherty* Department of Chemical Engineering, and Biomolecular Science and Engineering Program, University of California, Santa Barbara, Santa Barbara, California 93106

Fluorescence-activated cell sorting (FACS) was applied for quantitative screening of cDNA expression libraries in bacteria for rare fluorescent protein encoding cDNAs. Rare fluorescent cells, observed at a frequency of 1 in 200,000 bacteria in a cDNA expression library constructed from Astrangia lajollaensis, were detected, enriched, and purified by sorting, yielding three distinct green fluorescent proteins. Two of the isolated fluorescent proteins were found to be 2.5-fold brighter in whole cell fluorescence than the widely used and already optimized EGFP variant and possessed a novel cysteine-containing chromophore. FACS can possess significant advantages in the screening of cDNA libraries in bacteria, since desired genes may occur at low frequencies and possess unexpected properties. This strategy provides a highthroughput, quantitative approach for isolating fluorescent proteins from a more diverse range of organisms and should be extendable to proteins that are not intrinsically fluorescent with the use of available fluorescent indicators.

Introduction Autofluorescent proteins (AFPs) isolated from marine invertebrates have made an enormous impact on basic and applied biological research as molecular markers of gene expression and protein solubility, trafficking, and localization (1, 2). In addition, AFPs are increasingly used as molecular biosensor components and molecular recognition scaffolds (3, 4). Although a variety of fluorescent proteins have been isolated and engineered (5-11), the discovery of new fluorescent proteins remains of significant importance. Variants are needed with excitation and emission properties unrepresented by existing proteins, increased brightness and thermostabilty, reduced photobleaching, and properties more suitable for Fo¨rster resonance energy transfer (FRET) applications (2). Nearly all known AFPs have been isolated from reef Anthozoa species (12) using low-throughput and nonquantitative plate-based methods (13). Fluorescent tissue samples from the organism are homogenized, and cDNA is prepared using standard protocols. In some cases AFPencoding genes are preferentially amplified by a 3′-RACE method using degenerate primers based on known AFP sequences (7, 10, 13). The amplified genes or naı¨ve library is then transformed into E. coli, which are plated on solid media, and fluorescent colonies are observed under UV, blue, or green lights (6, 8, 14). This approach, however, may lead to undesirable bias in the diversity of fluorescent proteins that are eventually isolated, since mRNA stability, transcript frequency, excitation efficiency, and other factors could have adverse effects upon isolation. Also, these strategies may not be sufficient to observe interesting but dimly fluorescent proteins, as well as those that emit in the violet or near-IR regions of the spectrum. * To whom correspondence should be addressed. Tel: 805-8932610. Fax: 805-893-4731. Email: [email protected]. 10.1021/bp034308g CCC: $27.50

Given that fluorescence-activated cell sorting provides a powerful means to quantitatively identify and isolate cells on the basis of well-defined fluorescence properties (15), we applied FACS to isolate fluorescent proteins directly from cDNA expression libraries constructed in E. coli. Although flow cytometry is used frequently as a tool for analysis of AFP-expressing cells, FACS has been relatively underutilized thus far as a screening tool for the directed evolution of fluorescent proteins, an application for which it is ideally suited (16, 17). Furthermore, FACS has not been used previously to discover novel fluorescent proteins or, more generally, for screening heterologous cDNA libraries for proteins exhibiting specific desired functions.

Results and Discussion A cDNA library was constructed using mRNA isolated from the stony cup coral Astrangia lajollaensis, which displays bright green fluorescence under blue illumination. The library was constructed by first isolating total RNA, performing reverse transcription and amplification using the SMART cDNA PCR method (BD Biosciences), and then directional cloning into an expression vector employing the tightly regulated arabinose-inducible promoter from the araBAD operon (18). A tightly regulated expression vector was used to prevent unnecessary potential toxicity (as results from high-level expression of avGFP or DsRed) during library propagation, which could reduce the frequency of clones with adversely impacted growth rates (19). Prior to screening, the library was analyzed for the presence of fluorescent cells using flow cytometry. Fluorescent cells with varying fluorescence intensities occurred at a frequency on the order of 1 in 200,000 (Figure 1A). This frequency, though comparable, was lower than that observed in other studies (1:700-1:75,000) (6, 14), indicating that the isolation of AFP-expressing cells would have been intractable by plate screening. Consistent with

© 2004 American Chemical Society and American Institute of Chemical Engineers Published on Web 02/05/2004

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Figure 1. Flow cytometric screening of cDNA expression libraries in E. coli for fluorescent proteins. (A) Analysis of cDNA library expressing cell population prior to screening. (B) Cell population resulting from the sorting and regrowth of events occurring in gate R1 of 1A. (C) Population resulting from sorting and regrowth of the population occurring in R2 of 1B. (D) Population resulting from the sorting and regrowth of cells occurring in R3 of 1B.

this observation, the screening of 10,000 colonies using UV or blue-LED lamps did not reveal fluorescent clones. In the first cycle of library screening by FACS, all fluorescent events above background autofluorescence were sorted and amplified by overnight growth (Figure 1A). In the second cycle of sorting, the fluorescent cells fell into at least two distinct populations with unique fluorescence properties and were sorted separately (Figure 1B). Analysis of the two sorted samples the following day revealed a single population resulting from sorting highly fluorescent cells (Figure 1C) and two populations that resulted from sorting those cells with lower fluorescence (Figure 1D). Sequencing of the cDNA inserts confirmed the presence of three distinct species of green fluorescent clones, alajGFP1, 2, and 3 (using the naming convention suggested by Labas et al. (7)) (Figure 2). Clones alajGFP1 and alajGFP2 were 95% identical at the amino acid level; clone alajGFP3 was ∼66% identical to alajGFP1 or 2 and notably possessed a different chromophore tripeptide. The two clones with brighter, redshifted fluorescence possessed a chromophore containing cysteine (i.e., CYG), which had not been observed among isolated fluorescent proteins, whereas alajGFP3 possessed AYG at the corresponding positions. Pseudo-native PAGE (0.5% SDS loading buffer, 0.1% SDS running buffer) of unheated cell lysate soluble fractions revealed that alajGFP1 forms tetramers (data not shown). The oligomeric states of alajGFP2 and 3 were not determined; though soluble, the proteins were not fluorescent in the

gel, perhaps indicating lower SDS tolerance. The fluorescence spectra of alajGFP1 and 2 were identical, showing a single excitation peak with a maximum of 509 nm and an emission maximum of 517 nm. AlajGFP3 exhibited similar spectra with slightly blue-shifted excitation and emission maxima (494/504 nm). The three different open reading frames were recloned into the expression plasmid under the same ribosome binding site (RBS) for comparison of their brightness relative to EGFP (i.e., F64L, S65T mutant of GFP) (Figure 3). Such brightness measurements represent an aggregate measurement of quantum yield, extinction coefficient, maturation rate, and correctly folded expression level and, thus, provide a valuable indicator of functionality in whole-cell fluorescence assays (16). Significantly, both alajGFP1 and 2 exhibited approximately 2.5-fold brighter fluorescence in E. coli relative to EGFP, despite the fact that unlike EGFP they have not been optimized by mutagenesis (e.g., for efficient 488 nm excitation). After subcloning but without any changes to the coding sequence, the relative brightness increased by 5- to 50-fold, indicating that sequence of and distance from the native ribosomal binding site may reduce the probability of initially detecting bright fluorescent proteins that are weakly expressed. Alternative library construction methods, such as including an optimum RBS in the cloning vector and/or expressing the cDNA as a translational fusion protein, may increase the probability of successful isolation in such cases. The increased


Figure 2. Multiple sequence alignment of the three isolated fluorescent proteins, alajGFP1, 2, and 3. Residues identical among all three proteins are shaded black, residues conserved between two sequences are shaded gray, and gaps are represented by dashes. The chromophore tripeptide corresponds to positions 66-68.

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discrimination of multiple clones having differing fluorescence properties. The dim fluorescence of alajGFP2 and 3 expressed from their native RBS was such that they may not have been detected or discriminated in a platebased assay, even if occurring at a higher frequency. In addition, several reports describing both native and engineered AFPs indicate that fluorescence may take days or weeks to be detectable in colonies on plates, at which point viability may be compromised (8, 20). FACS likely would attenuate the screening time required in such cases. Second, improvements in laser technology and instrument optics have enabled the employment of low power diode lasers in a variety of interrogation wavelengths such that multiple excitation sources can be rapidly tested. The employment of multiple fluorescence detectors allows for emission ranges spanning the visible spectrum and potentially extending into the near-IR. Furthermore, multidimensional data plotting enables fine discrimination of clonal populations. Hence, it should be possible to identify AFPs, using alternative, efficient light sources (21, 22), more suitable for emerging biosensor applications. Finally and perhaps most importantly, this technique is truly high-throughput, allowing efficient processing of library sizes up to 109 in a few hours. Given this, it should be possible to screen large highly diverse genomic libraries derived from heterogeneous mixtures of nonculturable marine bacteria. Even relatively nonabundant genes could be represented in libraries that can be processed efficiently using FACS. FACS-based screening of cDNA expression libraries in bacteria provides a powerful new approach ideally suited for, but not limited to, fluorescent protein isolation. Since this method does not depend on knowledge of, or sequence identity to, other AFPs, such as in 3′-RACE-based methods (13), this methodology should enable efficient isolation of AFPs originating from a wider range of organisms and having properties tailored to specific biotechnology applications. Significantly, the work described here provides, to our knowledge, the first reported demonstration of FACS-based screening of a cDNA expression library in bacteria directly for functional proteins. Applied more generally, this methodology should be effective for screening cDNA and genomic DNA expression libraries from heterogeneous pools of organisms for desired protein activities (e.g., catalysis) because it enables even very rare clones to be detected and isolated.

Methods

Figure 3. Aggregate brightness measurement of alajGFP1, 2, and 3 relative to EGFP in E. coli, as measured using flow cytometry. Error bars represent ( SD of the mean green fluorescence of triplicate cultures. Light bars correspond to the cDNA clones isolated from the expression library and containing the native A. lajollaensis flanking sequences. Dark bars represent the clones generated by subcloning the coding region of each cDNA clone under the same RBS as the EGFP plasmid. (ND ) not determined).

brightness of alajGFP1 and 2 relative to EGFP should make them attractive alternatives to EGFP in some applications and could provide clues for rational redesign and evolution of existing AFPs. There are several advantages of AFP cloning using FACS, relative to traditional methods. First, sensitive FACS instrumentation enables the detection of even weakly fluorescent clones, as well as the quantitative

Bacterial Strains and Plasmids. E. coli strain MC1061 (23) was used for all cloning and expression studies. Expression vector pBKCm1 is based on pBAD18Cm (18) and contains two unique SfiI sites downstream of the araBAD promoter and flanking a kanamycin resistance (KanR) gene. EGFP expression vector pBEGFP contains a ribosome binding site and a gene for avGFP(F64L,S65T) inserted in pBKCm1 in place of the KanR cassette. Library Construction. Total RNA was extracted from ∼10 mg of Astrangia lajollaensis tissue that had been collected locally and stored at -20 °C in RNAlater solution (Ambion). The tissue was disrupted in an OMNI International homogenizer, and RNA was purified using an RNeasy spin column (QIAgen) according to the manufacturer’s protocol. The SMART PCR cDNA synthesis method (BD Biosciences), using modified oligonucleotides, was used for reverse transcription and amplification, following the supplier’s protocol except where noted. Briefly, 1 µg of total RNA was used in the

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first strand synthesis reaction with oligonucleotides PD242 (5′-AAGCAGTGGTATCAACGCAGAGTGGCCACGAAGGCCGGG-3′) and PD243 (5′-ATTCTAGAGGCCACCTTGGCCGACATG(T)30VN-3′), which have modified SfiI sites (underlined) that are compatible with directional cloning into plasmid pBKCm1. Following first strand synthesis, the cDNA was amplified by PCR in an MJ Research DYAD thermocycler for 20 cycles, using primers PD243 and 5′IIA (5′-AAGCAGTGGTATCAACGCAGAGT-3′). Oligonucleotides PD242 and PD243 were synthesized and RNase-free PAGE purified by Integrated DNA Technologies (Coralville, IA). Primer 5′IIA was obtained from BD Biosciences. Following amplification, the cDNA was purified on a QIAquick PCR Cleanup spin column (QIAgen) and digested with SfiI. The digested cDNA was electrophoresed on a 0.8% TAE agarose gel, and the fragments ∼500 bp to 8 kbp were excised from the gel and isolated using the QIAquick Gel Extraction kit (QIAgen). Digested, size-selected cDNA was then ligated to plasmid vector pBKCm1, which had been digested with SfiI and dephosphorylated with calf intestinal alkaline phosphatase. Ligated DNA was transformed into E. coli by electroporation, resulting in ∼107 independent transformants. Library Screening. For flow cytometric sorting, overnight cultures grown under repressing conditions (0.2% glucose) were diluted 100-fold into fresh LB medium containing 25 µg/mL chloramphenicol, grown with shaking to mid-log phase (ca. 2-3 h) at 37 °C, induced with 0.02% (w/v) L-arabinose, incubated an additional 3 h at 37 °C, and then shifted to 15 °C overnight. The following day, the cultures were diluted in PBS to ∼50,000 cells/µL, and ∼108 cells were processed on a Partec PAS III flow cytometer equipped with a 100 mW argon (488 nm) laser. All sorts were performed in recovery mode, where coincident events are not rejected. Green fluorescence was monitored through a 535/45 nm band-pass filter; red fluorescence was monitored through a 610/15 nm band-pass filter. All sorts were additionally gated on forward vs side light scatter parameters. Collected cells were grown in SOC medium supplemented with chloramphenicol (25 µg/mL) overnight at 37 °C. The second round of sorting was conducted as the first, but with 3 h of induction at 37 °C only. After overnight growth, cells were plated to LB agar containing 0.2% arabinose and 25 µg/mL chloramphenicol. Individual green colonies were identified from the plates by excitation with a blue (470 nm) LED lamp or by flow cytometry. Fluorimetric and Flow Cytometric Characterization of Isolated Clones. Fluorescence spectra were obtained from whole cells on a Cary Eclipse Spectrofluorometer. Pseudo-native PAGE to determine the oligomeric state was performed essentially as described (24) and imaged on a Storm 840 Imager (APBiotech) in blue fluorescence mode. Open reading frames were PCR amplified with primers containing a ribosome binding site and SfiI restriction sites compatible with those in pBKCm1. Resulting PCR products were subcloned into pBKCm1, and confirmed by DNA sequencing. For whole cell flow cytometric measurement of relative brightness, log-phase cultures were induced with 0.02% arabinose and cultured an additional 2.5 h at 37 °C. The fluorescence of 10,000 cells was measured using a Partec PAS-III, and mean fluorescence in the green channel (535/45 nm) was calculated using the FloMax software.

Acknowledgment We thank Daniel Morse, Shane Anderson, and James Weaver for helpful discussions and species confirmation,

Marco Mena for construction of pBKCm1 and pBEGFP, and Annalee Nguyen for carefully reading this manuscript. This work was supported in part by the National Institutes of Health Grant EB-00205. The cDNA sequence data reported in this paper have been submitted to the GenBank database under Accession Numbers AY508123, AY508124, AY508125.

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Accepted for publication January 7, 2004. BP034308G